Regulation of human serine-threonine protein kinase

ABSTRACT

Reagents which regulate human serine-threonine protein kinase and reagents which bind to human serine-threonine protein kinase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, cancer, CNS disorders, diabetes, and COPD.

[0001] This application claims the benefit of and incorporates byreference co-pending provisional applications Serial No. 60/308,118filed Jul. 30, 2001, Serial No. 60/314,112 filde Aug. 23, 2001, andco-pending provisional application Serial No. 60/240,073 filed Oct. 16,2000.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention relates to the area of enzyme regulation. Moreparticularly, the invention relates to the regulation of humanserine-threonine protein kinase.

BACKGROUND OF THE INVENTION

[0003] Intercellular signaling regulates a variety of importantbiological functions. For example, transforming growth factor type beta(TGF-β) regulates the proliferation and differentiation of a variety ofcell types binding to and activating cell surface receptors whichpossess serine/threonine kinase activity. Atfi et al. (Proc. Natl. Acad.Sci. U.S.A. 92, 12110-04, 1995) have shown that TGF-β activates a 78-kDaprotein (p78) serine/threonine kinase; the p78 kinase was activated onlyin cells for which TGF-β acts as a growth inhibitory factor. Because ofthe important functions of kinases such as p78, there is a need in theart to identify new kinases and methods of regulating these new kinasesfor therapeutic effects.

SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide reagents and methodsof regulating a human serine-threonine protein kinase. This and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

[0005] One embodiment of the invention is a serine-threonine proteinkinase polypeptide comprising an amino acid sequence selected from thegroup consisting of:

[0006] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 2;

[0007] the amino acid sequence shown in SEQ ID NO: 2;

[0008] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 17; and

[0009] the amino acid sequence shown in SEQ ID NO: 17.

[0010] Yet another embodiment of the invention is a method of screeningfor agents

[0011] which decrease extracellular matrix degradation. A test compoundis contacted with a serine-threonine protein kinase polypeptidecomprising an amino acid sequence selected from the group consisting of:

[0012] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 2; and

[0013] the amino acid sequence shown in SEQ ID NO: 2;

[0014] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 17; and

[0015] the amino acid sequence shown in SEQ ID NO: 17.

[0016] Binding between the test compound and the serine-threonineprotein kinase polypeptide is detected. A test compound which binds tothe serine-threonine protein kinase polypeptide is thereby identified asa potential agent for decreasing extracellular matrix degradation. Theagent can work by decreasing the activity of the serine-threonineprotein kinase.

[0017] Another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a polynucleotide encoding a serine-threonine proteinkinase polypeptide, wherein the polynucleotide comprises a nucleotidesequence selected from the group consisting of:

[0018] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 1; and

[0019] the nucleotide sequence shown in SEQ ID NO: 1.

[0020] Binding of the test compound to the polynucleotide is detected. Atest compound which binds to the polynucleotide is identified as apotential agent for decreasing extracellular matrix degradation. Theagent can work by decreasing the amount of the serine-threonine proteinkinase through interacting with the serine-threonine protein kinasemRNA.

[0021] Another embodiment of the invention is a method of screening foragents which regulate extracellular matrix degradation. A test compoundis contacted with a serine-threonine protein kinase polypeptidecomprising an amino acid sequence selected from the group consisting of:

[0022] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 2; and

[0023] the amino acid sequence shown in SEQ ID NO: 2;

[0024] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 17; and

[0025] the amino acid sequence shown in SEQ ID NO: 17.

[0026] A serine-threonine protein kinase activity of the polypeptide isdetected. A test compound which increases serine-threonine proteinkinase activity of the polypeptide relative to serine-threonine proteinkinase activity in the absence of the test compound is therebyidentified as a potential agent for increasing extracellular matrixdegradation. A test compound which decreases serine-threonine proteinkinase activity of the polypeptide relative to serine-threonine proteinkinase activity in the absence of the test compound is therebyidentified as a potential agent for decreasing extracellular matrixdegradation.

[0027] Even another embodiment of the invention is a method of screeningfor agents which decrease extracellular matrix degradation. A testcompound is contacted with a serine-threonine protein kinase product ofa polynucleotide which comprises a nucleotide sequence selected from thegroup consisting of:

[0028] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 1; and

[0029] the nucleotide sequence shown in SEQ ID NO: 1.

[0030] Binding of the test compound to the serine-threonine proteinkinase product is detected. A test compound which binds to theserine-threonine protein kinase product is thereby identified as apotential agent for decreasing extracellular matrix degradation.

[0031] Still another embodiment of the invention is a method of reducingextracellular matrix degradation. A cell is contacted with a reagentwhich specifically binds to a polynucleotide encoding a serine-threonineprotein kinase polypeptide or the product encoded by the polynucleotide,wherein the polynucleotide comprises a nucleotide sequence selected fromthe group consisting of:

[0032] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 1; and

[0033] the nucleotide sequence shown in SEQ ID NO: 1.

[0034] Serine-threonine protein kinase activity in the cell is therebydecreased.

[0035] The invention thus provides a human serine-threonine proteinkinase which can be used to identify test compounds which may act, forexample, as activators or inhibitors at the enzyme's active site. Humanserine-threonine protein kinase and fragments thereof also are useful inraising specific antibodies which can block the enzyme and effectivelyreduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide.

[0037]FIG. 2 shows the amino acid sequence deduced from the DNA-sequenceof FIG. 1 (SEQ ID NO:2).

[0038]FIG. 3 shows the amino acid sequence of the protein identified bySwissProt Accession No. 070405 (SEQ ID NO:3).

[0039]FIG. 4 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:1).

[0040]FIG. 5 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:4).

[0041]FIG. 6 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:5).

[0042]FIG. 7 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:6).

[0043]FIG. 8 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:7).

[0044]FIG. 9 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:8).

[0045]FIG. 10 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:9).

[0046]FIG. 11 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:10).

[0047]FIG. 12 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:11).

[0048]FIG. 13 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:12).

[0049]FIG. 14 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:13).

[0050]FIG. 15 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO: 14).

[0051]FIG. 16 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:15).

[0052]FIG. 17 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:16).

[0053]FIG. 18 shows the amino acid sequence of a serine-threonineprotein kinase Polypeptide (SEQ ID NO:17).

[0054]FIG. 19 shows the DNA-sequence encoding a serine-threonine proteinkinase Polypeptide (SEQ ID NO:18).

[0055]FIG. 20 shows the BLASTP alignment of human serine-threonineprotein kinase (SEQ ID NO:2) with the protein identified with SwissProtAccession No. P07405 (SEQ ID NO:3).

[0056]FIG. 21 shows the HMMPFAM—alignment of 127 (SEQ ID NO:2) againstpfam|hmm|pkinase.

[0057]FIG. 22 shows the human serine/threonine kinase relativeexpression in cancer tissues.

[0058]FIGS. 23a and b shows the human serine/threonine kinase relativemRNA expression in tissues relevant for obesity and diabetes.

[0059]FIG. 24 shows the BLASTP—alignment of 127_V3_protein againstaageneseq|AAB65692|AAB65692.

[0060]FIG. 25 shows the TBLASTN—alignment of 127_V3_protein againstnageneseq|AAD03991|AAD03991.

[0061]FIG. 26 shows the BLASTP—alignment of 127_V3_protein againstaageneseq|AAE00665|AAE00665.

[0062]FIG. 27 shows the BLASTP—alignment of 127_V3_protein againstaageneseq|AAB41873|AAB41873.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The invention relates to an isolated polynucleotide encoding aserine-threonine protein kinase polypeptide and being selected from thegroup consisting of:

[0064] a) a polynucleotide encoding a serine-threonine protein kinasepolypeptide comprising an amino acid sequence selected from the groupconsisting of:

[0065] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 2; and

[0066] the amino acid sequence shown in SEQ ID NO: 2;

[0067] amino acid sequences which are at least about 30% identical tothe amino acid sequence shown in SEQ ID NO: 17; and

[0068] he amino acid sequence shown in SEQ ID NO: 17.

[0069] b) a polynucleotide comprising the sequence of SEQ ID NO: 1 or16;

[0070] c) a polynucleotide which hybridizes under stringent conditionsto a polynucleotide specified in (a) and (b);

[0071] d) a polynucleotide the sequence of which deviates from thepolynucleotide sequences specified in (a) to (c) due to the degenerationof the genetic code; and

[0072] e) a polynucleotide which represents a fragment, derivative orallelic variation of a polynucleotide sequence specified in (a) to (d).

[0073] Furthermore, it has been discovered by the present applicant thata novel serine-threonine protein kinase, particularly a humanserine-threonine protein kinase, is a discovery of the presentinvention. Human serine-threonine protein kinase comprises the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:17. A coding sequencefor human serine-threonine protein kinase is shown in SEQ ID NO:1 andSEQ ID NO:16. Related ESTs (SEQ ID NOS:5-16) are expressed in highlyproliferating cells, such as pooled germ cell tumors, prostate, Tlymphocytes, germinal center B cells, testis, and renal proximal tubuleepithelial cells.

[0074] Human serine-threonine protein kinase is 29% identical over 287amino acids to the mouse protein identified with SwissProt Accession No.O70405 (SEQ ID NO:3) and annotated as “SERINE/THREONINE-PROTEIN KINASEULK1” (FIG. 20).

[0075] Human serine-threonine protein kinase is 99% identical over 437amino acids to human protein tyrosine kinase receptor cDNA from cloneHTAEV17 (FIG. 25).

[0076] Human serine-threonine protein kinase of the invention isexpected to be useful for the same purposes as previously identifiedserine-threonine kinase enzymes. Human serine-threonine protein kinaseis believed to be useful in therapeutic methods to treat disorders suchas cancer, CNS disorders, diabetes, and COPD. Human serine-threonineprotein kinase also can be used to screen for human serine-threonineprotein kinase activators and inhibitors.

[0077] Polypeptides

[0078] Human serine-threonine protein kinase polypeptides according tothe invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, or 542 contiguous amino acids selected from the amino acidsequence shown in SEQ ID NO:2 or 17 or a biologically active variantthereof, as defined below. Human serine-threonine protein kinasepolypeptides according to the invention comprise at least 6, 10, 15, 20,25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, or 580 contiguous amino acidsselected from the amino acid sequence shown in SEQ ID NO:2 or 17 or abiologically active variant thereof, as defined below. Aserine-threonine protein kinase polypeptide of the invention thereforecan be a portion of a serine-threonine protein kinase protein, afull-length serine-threonine protein kinase protein, or a fusion proteincomprising all or a portion of a serine-threonine protein kinaseprotein.

[0079] Biologically Active Variants

[0080] Human serine-threonine protein kinase polypeptide variants whichare biologically active, e.g., retain a kinase activity, also areserine-threonine protein kinase polypeptides. Preferably, naturally ornon-naturally occurring serine-threonine protein kinase polypeptidevariants have amino acid sequences which are at least about 30, 35, 40,45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or98% identical to the amino acid sequence shown in SEQ ID NO:2 or 17 or afragment thereof. Percent identity between a putative serine-threonineprotein kinase polypeptide variant and an amino acid sequence of SEQ IDNO:2 or 17 is determined using the Blast2 alignment program (Blosum62,Expect 10, standard genetic codes).

[0081] Variations in percent identity can be due, for example, to aminoacid substitutions, insertions, or deletions. Amino acid substitutionsare defined as one for one amino acid replacements. They areconservative in nature when the substituted amino acid has similarstructural and/or chemical properties. Examples of conservativereplacements are substitution of a leucine with an isoleucine or valine,an aspartate with a glutamate, or a threonine with a serine.

[0082] Amino acid insertions or deletions are changes to or within anamino acid sequence. They typically fall in the range of about 1 to 5amino acids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of a serine-threonine protein kinase polypeptidecan be found using computer programs well known in the art, such asDNASTAR software. Whether an amino acid change results in a biologicallyactive serine-threonine protein kinase polypeptide can readily bedetermined by assaying for kinase activity, as described for example, inExample 4.

[0083] Fusion Proteins

[0084] Fusion proteins are useful for generating antibodies againstserine-threonine protein kinase polypeptide amino acid sequences and foruse in various assay systems. For example, fusion proteins can be usedto identify proteins which interact with portions of a serine-threonineprotein kinase polypeptide. Protein affinity chromatography orlibrary-based assays for protein-protein interactions, such as the yeasttwo-hybrid or phage display systems, can be used for this purpose. Suchmethods are well known in the art and also can be used as drug screens.

[0085] A serine-threonine protein kinase polypeptide fusion proteincomprises two polypeptide segments fused together by means of a peptidebond. The first polypeptide segment comprises at least 6, 10, 15, 20,25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, or 542 contiguous amino acids of SEQ IDNO:2 or 17 or of a biologically active variant, such as those describedabove. A serine-threonine protein kinase polypeptide fusion proteincomprises two polypeptide segments fused together by means of a peptidebond. The first polypeptide segment comprises at least 6, 10, 15, 20,25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, or 580 contiguous amino acids of SEQID NO:2 or 17 or of a biologically active variant, such as thosedescribed above. The first polypeptide segment also can comprisefull-length serine-threonine protein kinase protein.

[0086] The second polypeptide segment can be a full-length protein or aprotein fragment. Proteins commonly used in fusion protein constructioninclude β-galactosidase, β-glucuronidase, green fluorescent protein(GFP), autofluorescent proteins, including blue fluorescent protein(BFP), glutathione-S-transferase (GST), luciferase, horseradishperoxidase (HRP), and chloramphenicol acetyltransferase (CAT).Additionally, epitope tags are used in fusion protein constructions,including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSVG tags, and thioredoxin (Trx) tags. Other fusionconstructions can include maltose binding protein (MBP), S-tag, Lex aDNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. A fusion protein alsocan be engineered to contain a cleavage site located between theserine-threonine protein kinase polypeptide-encoding sequence and theheterologous protein sequence, so that the serine-threonine proteinkinase polypeptide can be cleaved and purified away from theheterologous moiety.

[0087] A fusion protein can be synthesized chemically, as is known inthe art. Preferably, a fusion protein is produced by covalently linkingtwo polypeptide segments or by standard procedures in the art ofmolecular biology. Recombinant DNA methods can be used to prepare fusionproteins, for example, by making a DNA construct which comprises codingsequences selected from SEQ ID NO:1 or SEQ ID NO:16 in proper readingframe with nucleotides encoding the second polypeptide segment andexpressing the DNA construct in a host cell, as is known in the art.Many kits for constructing fusion proteins are available from companiessuch as Promega Corporation (Madison, Wis.), Stratagene (La Jolla,Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology(Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown,Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0088] Identification of Species Homologs

[0089] Species homologs of human serine-threonine protein kinasepolypeptide can be obtained using serine-threonine protein kinasepolypeptide polynucleotides (described below) to make suitable probes orprimers for screening cDNA expression libraries from other species, suchas mice, monkeys, or yeast, identifying cDNAs which encode homologs ofserine-threonine protein kinase polypeptide, and expressing the cDNAs asis known in the art.

[0090] Polynucleotides

[0091] A serine-threonine protein kinase polynucleotide can be single-or double-stranded and comprises a coding sequence or the complement ofa coding sequence for a serine-threonine protein kinase polypeptide. Acoding sequence for human serine-threonine protein kinase is shown inSEQ ID NO:1 and 16. This sequence is contained within a longer genomicsequence shown in SEQ ID NO:4.

[0092] Degenerate nucleotide sequences encoding human serine-threonineprotein kinase polypeptides, as well as homologous nucleotide sequenceswhich are at least about 50, 55, 60, 65, 70, preferably about 75, 90,96, or 98% identical to the nucleotide sequence shown in SEQ ID NO:1 or16 or its complement also are serine-threonine protein kinasepolynucleotides. Percent sequence identity between the sequences of twopolynucleotides is determined using computer programs such as ALIGNwhich employ the FASTA algorithm, using an affine gap search with a gapopen penalty of −12 and a gap extension penalty of −2. Complementary DNA(cDNA) molecules, species homologs, and variants of serine-threonineprotein kinase polynucleotides which encode biologically activeserine-threonine protein kinase polypeptides also are serine-threonineprotein kinase polynucleotides.

[0093] Identification of Polynucleotide Variants and Homologs

[0094] Variants and homologs of the serine-threonine protein kinasepolynucleotides described above also are serine-threonine protein kinasepolynucleotides. Typically, homologous serine-threonine protein kinasepolynucleotide sequences can be identified by hybridization of candidatepolynucleotides to known serine-threonine protein kinase polynucleotidesunder stringent conditions, as is known in the art. For example, usingthe following wash conditions—2X SSC (0.3 M NaCl, 0.03 M sodium citrate,pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC,0.1% SDS, 50° C. once, 30 minutes; then 2X SSC, room temperature twice,10 minutes each—homologous sequences can be identified which contain atmost about 25-30% basepair mismatches. More preferably, homologousnucleic acid strands contain 15-25% basepair mismatches, even morepreferably 5-15% basepair mismatches.

[0095] Species homologs of the serine-threonine protein kinasepolynucleotides disclosed herein also can be identified by makingsuitable probes or primers and screening cDNA expression libraries fromother species, such as mice, monkeys, or yeast. Human variants ofserine-threonine protein kinase polynucleotides can be identified, forexample, by screening human cDNA expression libraries. It is well knownthat the T_(m) of a double-stranded DNA decreases by 1-1.5° C. withevery 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123(1973). Variants of human serine-threonine protein kinasepolynucleotides or serine-threonine protein kinase polynucleotides ofother species can therefore be identified by hybridizing a putativehomologous serine-threonine protein kinase polynucleotide with apolynucleotide having a nucleotide sequence of SEQ ID NO:1 or 16 or thecomplement thereof to form a test hybrid. The melting temperature of thetest hybrid is compared with the melting temperature of a hybridcomprising polynucleotides having perfectly complementary nucleotidesequences, and the number or percent of basepair mismatches within thetest hybrid is calculated.

[0096] Nucleotide sequences which hybridize to serine-threonine proteinkinase polynucleotides or their complements following stringenthybridization and/or wash conditions also are serine-threonine proteinkinase polynucleotides. Stringent wash conditions are well known andunderstood in the art and are disclosed, for example, in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages9.50-9.51.

[0097] Typically, for stringent hybridization conditions a combinationof temperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a serine-threonine protein kinasepolynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or SEQID NO:16 or the complement thereof and a polynucleotide sequence whichis at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or98% identical to one of those nucleotide sequences can be calculated,for example, using the equation of Bolton and McCarthy, Proc. Natl.Acad. Sci. U.S.A. 48, 1390 (1962):

T _(m)=81.5° C.−16.6(log₁₀ [Na ⁺])+0.41(%G+C)−0.63(%formamide)−600/l),

[0098] where l=the length of the hybrid in basepairs.

[0099] Stringent wash conditions include, for example, 4X SSC at 65° C.,or 50% formamide, 4X SSC at 42° C., or 0.5X SSC, 0.1% SDS at 65° C.Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

[0100] Preparation of Polynucleotides

[0101] A serine-threonine protein kinase polynucleotide can be isolatedfree of other cellular components such as membrane components, proteins,and lipids. Polynucleotides can be made by a cell and isolated usingstandard nucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated serine-threonine proteinkinase polynucleotides. For example, restriction enzymes and probes canbe used to isolate polynucleotide fragments which comprisesserine-threonine kinase nucleotide sequences. Isolated polynucleotidesare in preparations which are free or at least 70, 80, or 90% free ofother molecules.

[0102] Human serine-threonine protein kinase cDNA molecules can be madewith standard molecular biology techniques, using serine-threonineprotein kinase mRNA as a template. Human serine-threonine protein kinasecDNA molecules can thereafter be replicated using molecular biologytechniques known in the art and disclosed in manuals such as Sambrook etal. (1989). An amplification technique, such as PCR, can be used toobtain additional copies of polynucleotides of the invention, usingeither human genomic DNA or cDNA as a template.

[0103] Alternatively, synthetic chemistry techniques can be used tosynthesizes serine-threonine protein kinase polynucleotides. Thedegeneracy of the genetic code allows alternate nucleotide sequences tobe synthesized which will encode a serine-threonine protein kinasepolypeptide having, for example, an amino acid sequence shown in SEQ IDNO:2 or SEQ ID NO:17 or a biologically active variant thereof.

[0104] Extending Polynucleotides

[0105] Various PCR-based methods can be used to extend the nucleic acidsequences disclosed herein to detect upstream sequences such aspromoters and regulatory elements. For example, restriction-site PCRuses universal primers to retrieve unknown sequence adjacent to a knownlocus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA isfirst amplified in the presence of a primer to a linker sequence and aprimer specific to the known region. The amplified sequences are thensubjected to a second round of PCR with the same linker primer andanother specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase andsequenced using reverse transcriptase.

[0106] Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16, 8186, 1988). Primers can be designed using commerciallyavailable software, such as OLIGO 4.06 Primer Analysis software(National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0107] Another method which can be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom et al., PCR MethodsApplic. 1, 111-119, 1991). In this method, multiple restriction enzymedigestions and ligations also can be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0108] Another method which can be used to retrieve unknown sequences isthat of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991).Additionally, PCR, nested primers, and PROMOTERFINDER libraries(CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH,Palo Alto, Calif.). This process avoids the need to screen libraries andis useful in finding intron/exon junctions.

[0109] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs.Randomly-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariescan be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0110] Commercially available capillary electrophoresis systems can beused to analyze the size or confirm the nucleotide sequence of PCR orsequencing products. For example, capillary sequencing can employflowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity can be converted to electrical signalusing appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR,Perkin Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display can be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0111] Obtaining Polypeptides

[0112] Human serine-threonine protein kinase polypeptides can beobtained, for example, by purification from human cells, by expressionof serine-threonine protein kinase polynucleotides, or by directchemical synthesis.

[0113] Protein Purification

[0114] Human serine-threonine protein kinase polypeptides can bepurified from any cell which expresses the enzyme, including host cellswhich have been transfected with serine-threonine protein kinaseexpression constructs. A purified serine-threonine protein kinasepolypeptide is separated from other compounds which normally associatewith the serine-threonine protein kinase polypeptide in the cell, suchas certain proteins, carbohydrates, or lipids, using methods well-knownin the art. Such methods include, but are not limited to, size exclusionchromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified serine-threonine proteinkinase polypeptides is at least 80% pure; preferably, the preparationsare 90%, 95%, or 99% pure. Purity of the preparations can be assessed byany means known in the art, such as SDS-polyacrylamide gelelectrophoresis.

[0115] Expression of Polynucleotides

[0116] To express a serine-threonine protein kinase polynucleotide, thepolynucleotide can be inserted into an expression vector which containsthe necessary elements for the transcription and translation of theinserted coding sequence. Methods which are well known to those skilledin the art can be used to construct expression vectors containingsequences encoding serine-threonine protein kinase polypeptides andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook et al. (1989) and in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1989.

[0117] A variety of expression vector/host systems can be utilized tocontain and express sequences encoding a serine-threonine protein kinasepolypeptide. These include, but are not limited to, microorganisms, suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors, insect cell systems infected with virus expression vectors(e.g., baculovirus), plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids), or animal cell systems.

[0118] The control elements or regulatory sequences are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like canbe used. The baculovirus polyhedrin promoter can be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO, and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) can be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of a nucleotide sequenceencoding a serine-threonine protein kinase polypeptide, vectors based onSV40 or EBV can be used with an appropriate selectable marker.

[0119] Bacterial and Yeast Expression Systems

[0120] In bacterial systems, a number of expression vectors can beselected depending upon the use intended for the serine-threonineprotein kinase polypeptide. For example, when a large quantity of aserine-threonine protein kinase polypeptide is needed for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified can be used. Such vectors include,but are not limited to, multifunctional E. coli cloning and expressionvectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, asequence encoding the serine-threonine protein kinase polypeptide can beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also canbe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems can be designed to includeheparin, thrombin, or factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0121] In the yeast Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al.(1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.

[0122] Plant and Insect Expression Systems

[0123] If plant expression vectors are used, the expression of sequencesencoding serine-threonine protein kinase polypeptides can be driven byany of a number of promoters. For example, viral promoters such as the35S and 19S promoters of CaMV can be used alone or in combination withthe omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311,1987). Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3,1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter etal., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs canbe introduced into plant cells by direct DNA transformation or bypathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (e.g., Hobbs or Murray, in MCGRAWHILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y.,pp. 191-196, 1992).

[0124] An insect system also can be used to express a serine-threonineprotein kinase polypeptide. For example, in one such system Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. Sequences encoding serine-threonine protein kinase polypeptidescan be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of serine-threonine protein kinase polypeptideswill render the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses can then be used to infectS. frugiperda cells or Trichoplusia larvae in which serine-threonineprotein kinase polypeptides can be expressed (Engelhard et al., Proc.Nat. Acad. Sci. 91, 3224-3227, 1994).

[0125] Mammalian Expression Systems

[0126] A number of viral-based expression systems can be used to expressserine-threonine protein kinase polypeptides in mammalian host cells.For example, if an adenovirus is used as an expression vector, sequencesencoding serine-threonine protein kinase polypeptides can be ligatedinto an adenovirus transcription/translation complex comprising the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome can be used to obtain a viable viruswhich is capable of expressing a serine-threonine protein kinasepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such asthe Rous sarcoma virus (RSV) enhancer, can be used to increaseexpression in mammalian host cells.

[0127] Human artificial chromosomes (HACs) also can be used to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6M to 10M are constructed and delivered to cells viaconventional delivery methods (e.g., liposomes, polycationic aminopolymers, or vesicles).

[0128] Specific initiation signals also can be used to achieve moreefficient translation of sequences encoding serine-threonine proteinkinase polypeptides. Such signals include the ATG initiation codon andadjacent sequences. In cases where sequences encoding a serine-threonineprotein kinase polypeptide, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals (including the ATG initiationcodon) should be provided. The initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons can be of various origins,both natural and synthetic. The efficiency of expression can be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used (see Scharf et al., Results Probl. CellDiffer. 20, 125-162, 1994).

[0129] Host Cells

[0130] A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedserine-threonine protein kinase polypeptide in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va., 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

[0131] Stable expression is preferred for long-term, high-yieldproduction of recombinant proteins. For example, cell lines which stablyexpress serine-threonine protein kinase polypeptides can be transformedusing expression vectors which can contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediumbefore they are switched to a selective medium. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced serine-threonine protein kinase sequences. Resistant clonesof stably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell type. See, for example, ANIMAL CELLCULTURE, R. I. Freshney, ed., 1986.

[0132] Any number of selection systems can be used to recovertransformed cell lines.

[0133] These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adeninephosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980),npt confers resistance to the aminoglycosides, neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and patconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murray, 1992, supra). Additionalselectable genes have been described. For example, trpB allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci 85, 8047-51, 1988). Visible markers such as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, can be used to identify transformants and to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131,1995).

[0134] Detecting Expression

[0135] Although the presence of marker gene expression suggests that theserine-threonine protein kinase polynucleotide is also present, itspresence and expression may need to be confirmed. For example, if asequence encoding a serine-threonine protein kinase polypeptide isinserted within a marker gene sequence, transformed cells containingsequences which encode a serine-threonine protein kinase polypeptide canbe identified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with a sequence encoding aserine-threonine protein kinase polypeptide under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the serine-threonineprotein kinase polynucleotide.

[0136] Alternatively, host cells which contain a serine-threonineprotein kinase polynucleotide and which express a serine-threonineprotein kinase polypeptide can be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, orchip-based technologies for the detection and/or quantification ofnucleic acid or protein. For example, the presence of a polynucleotidesequence encoding a serine-threonine protein kinase polypeptide can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding aserine-threonine protein kinase polypeptide. Nucleic acidamplification-based assays involve the use of oligonucleotides selectedfrom sequences encoding a serine-threonine protein kinase polypeptide todetect transformants which contain a serine-threonine protein kinasepolynucleotide.

[0137] A variety of protocols for detecting and measuring the expressionof a serine-threonine protein kinase polypeptide, using eitherpolyclonal or monoclonal antibodies specific for the polypeptide, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting(FACS). A two-site, monoclonal-based immunoassay using monoclonalantibodies reactive to two non-interfering epitopes on aserine-threonine protein kinase polypeptide can be used, or acompetitive binding assay can be employed. These and other assays aredescribed in Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL,APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158,1211-1216, 1983).

[0138] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingserine-threonine protein kinase polypeptides include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences encoding a serine-threonine proteinkinase polypeptide can be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, and fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0139] Expression and Purification of Polypeptides

[0140] Host cells transformed with nucleotide sequences encoding aserine-threonine protein kinase polypeptide can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. As will be understood by those of skill in the art,expression vectors containing polynucleotides which encodeserine-threonine protein kinase polypeptides can be designed to containsignal sequences which direct secretion of soluble serine-threonineprotein kinase polypeptides through a prokaryotic or eukaryotic cellmembrane or which direct the membrane insertion of membrane-boundserine-threonine protein kinase polypeptide.

[0141] As discussed above, other constructions can be used to join asequence encoding a serine-threonine protein kinase polypeptide to anucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Inclusion ofcleavable linker sequences such as those specific for Factor Xa orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the serine-threonine protein kinase polypeptide also can beused to facilitate purification. One such expression vector provides forexpression of a fusion protein containing a serine-threonine proteinkinase polypeptide and 6 histidine residues preceding a thioredoxin oran enterokinase cleavage site. The histidine residues facilitatepurification by IMAC (immobilized metal ion affinity chromatography, asdescribed in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), whilethe enterokinase cleavage site provides a means for purifying theserine-threonine protein kinase polypeptide from the fusion protein.Vectors which contain fusion proteins are disclosed in Kroll et al., DNACell Biol. 12, 441-453, 1993.

[0142] Chemical Synthesis

[0143] Sequences encoding a serine-threonine protein kinase polypeptidecan be synthesized, in whole or in part, using chemical methods wellknown in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser.215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a serine-threonine protein kinase polypeptide itself canbe produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Robergeet al., Science 269, 202-204, 1995). Protein synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Optionally, fragments of serine-threonine protein kinasepolypeptides can be separately synthesized and combined using chemicalmethods to produce a full-length molecule.

[0144] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, W H Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic serine-threonineprotein kinase polypeptide can be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton,supra). Additionally, any portion of the amino acid sequence of theserine-threonine protein kinase polypeptide can be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins to produce a variant polypeptide or a fusion protein.

[0145] Production of Altered Polypeptides

[0146] As will be understood by those of skill in the art, it may beadvantageous to produce serine-threonine protein kinasepolypeptide-encoding nucleotide sequences possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0147] The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter serine-threonine proteinkinase polypeptide-encoding sequences for a variety of reasons,including but not limited to, alterations which modify the cloning,processing, and/or expression of the polypeptide or mRNA product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides can be used to engineer the nucleotidesequences. For example, site-directed mutagenesis can be used to insertnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations, and so forth.

[0148] Antibodies

[0149] Any type of antibody known in the art can be generated to bindspecifically to an epitope of a serine-threonine protein kinasepolypeptide. “Antibody” as used herein includes intact immunoglobulinmolecules, as well as fragments thereof, such as Fab, F(ab′)₂, and Fv,which are capable of binding an epitope of a serine-threonine proteinkinase polypeptide. Typically, at least 6, 8, 10, or 12 contiguous aminoacids are required to form an epitope. However, epitopes which involvenon-contiguous amino acids may require more, e.g., at least 15, 25, or50 amino acids.

[0150] An antibody which specifically binds to an epitope of aserine-threonine protein kinase polypeptide can be used therapeutically,as well as in immunochemical assays, such as Western blots, ELISAs,radioimmunoassays, immunohistochemical assays, immunoprecipitations, orother immunochemical assays known in the art. Various immunoassays canbe used to identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody whichspecifically binds to the immunogen.

[0151] Typically, an antibody which specifically binds to aserine-threonine protein kinase polypeptide provides a detection signalat least 5-, 10-, or 20-fold higher than a detection signal providedwith other proteins when used in an immunochemical assay. Preferably,antibodies which specifically bind to serine-threonine kinasepolypeptides do not detect other proteins in immunochemical assays andcan immunoprecipitate a serine-threonine protein kinase polypeptide fromsolution.

[0152] Human serine-threonine protein kinase polypeptides can be used toimmunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, orhuman, to produce polyclonal antibodies. If desired, a serine-threonineprotein kinase polypeptide can be conjugated to a carrier protein, suchas bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.Depending on the host species, various adjuvants can be used to increasethe immunological response. Such adjuvants include, but are not limitedto, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol). Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially useful.

[0153] Monoclonal antibodies which specifically bind to aserine-threonine protein kinase polypeptide can be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These techniques include, but are notlimited to, the hybridoma technique, the human B-cell hybridomatechnique, and the EBV-hybridoma technique (Kohler et al., Nature 256,495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Coteet al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al., Mol.Cell Biol. 62, 109-120, 1984).

[0154] In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.Alternatively, humanized antibodies can be produced using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto a serine-threonine protein kinase polypeptide can contain antigenbinding sites which are either partially or fully humanized, asdisclosed in U.S. Pat. No. 5,565,332.

[0155] Alternatively, techniques described for the production of singlechain antibodies can be adapted using methods known in the art toproduce single chain antibodies which specifically bind toserine-threonine protein kinase polypeptides. Antibodies with relatedspecificity, but of distinct idiotypic composition, can be generated bychain shuffling from random combinatorial immunoglobin libraries(Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

[0156] Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

[0157] A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91).

[0158] Antibodies which specifically bind to serine-threonine proteinkinase polypeptides also can be produced by inducing in vivo productionin the lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989;Winter et al., Nature 349, 293-299, 1991).

[0159] Other types of antibodies can be constructed and usedtherapeutically in methods of the invention. For example, chimericantibodies can be constructed as disclosed in WO 93/03151. Bindingproteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared.

[0160] Antibodies according to the invention can be purified by methodswell known in the art. For example, antibodies can be affinity purifiedby passage over a column to which a serine-threonine protein kinasepolypeptide is bound. The bound antibodies can then be eluted from thecolumn using a buffer with a high salt concentration.

[0161] Antisense Oligonucleotides

[0162] Antisense oligonucleotides are nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofserine-threonine protein kinase gene products in the cell.

[0163] Antisense oligonucleotides can be deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages suchalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994;Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev.90, 543-583, 1990.

[0164] Modifications of serine-threonine protein kinase gene expressioncan be obtained by designing antisense oligonucleotides which will formduplexes to the control, 5′, or regulatory regions of theserine-threonine protein kinase gene. Oligonucleotides derived from thetranscription initiation site, e.g., between positions −10 and +10 fromthe start site, are preferred. Similarly, inhibition can be achievedusing “triple helix” base-pairing methodology. Triple helix pairing isuseful because it causes inhibition of the ability of the double helixto open sufficiently for the binding of polymerases, transcriptionfactors, or chaperons. Therapeutic advances using triplex DNA have beendescribed in the literature (e.g., Gee et al., in Huber & Carr,MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco,N.Y., 1994). An antisense oligonucleotide also can be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes.

[0165] Precise complementarity is not required for successful complexformation between an antisense oligonucleotide and the complementarysequence of a serine-threonine protein kinase polynucleotide. Antisenseoligonucleotides which comprise, for example, 2, 3, 4, or 5 or morestretches of contiguous nucleotides which are precisely complementary toa serine-threonine protein kinase polynucleotide, each separated by astretch of contiguous nucleotides which are not complementary toadjacent serine-threonine protein kinase nucleotides, can providesufficient targeting specificity for serine-threonine protein kinasemRNA. Preferably, each stretch of complementary contiguous nucleotidesis at least 4, 5, 6, 7, or 8 or more nucleotides in length.Non-complementary intervening sequences are preferably 1, 2, 3, or 4nucleotides in length. One skilled in the art can easily use thecalculated melting point of an antisense-sense pair to determine thedegree of mismatching which will be tolerated between a particularantisense oligonucleotide and a particular serine-threonine proteinkinase polynucleotide sequence.

[0166] Antisense oligonucleotides can be modified without affectingtheir ability to hybridize to a serine-threonine protein kinasepolynucleotide. These modifications can be internal or at one or bothends of the antisense molecule. For example, internucleoside phosphatelinkages can be modified by adding cholesteryl or diamine moieties withvarying numbers of carbon residues between the amino groups and terminalribose. Modified bases and/or sugars, such as arabinose instead ofribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxylgroup or the 5′ phosphate group are substituted, also can be employed ina modified antisense oligonucleotide. These modified oligonucleotidescan be prepared by methods well known in the art. See, e.g., Agrawal etal., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev.90, 543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542,1987.

[0167] Ribozymes

[0168] Ribozymes are RNA molecules with catalytic activity. See, e.g.,Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59,543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture& Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

[0169] The coding sequence of a serine-threonine protein kinasepolynucleotide can be used to generate ribozymes which will specificallybind to mRNA transcribed from the serine-threonine protein kinasepolynucleotide. Methods of designing and constructing ribozymes whichcan cleave other RNA molecules in trans in a highly sequence specificmanner have been developed and described in the art (see Haseloff et al.Nature 334, 585-591, 1988). For example, the cleavage activity ofribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the target (see, for example, Gerlach etal., EP 321,201).

[0170] Specific ribozyme cleavage sites within a serine-threonineprotein kinase RNA target can be identified by scanning the targetmolecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetarget RNA containing the cleavage site can be evaluated for secondarystructural features which may render the target inoperable. Suitabilityof candidate serine-threonine protein kinase RNA targets also can beevaluated by testing accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays. Longercomplementary sequences can be used to increase the affinity of thehybridization sequence for the target. The hybridizing and cleavageregions of the ribozyme can be integrally related such that uponhybridizing to the target RNA through the complementary regions, thecatalytic region of the ribozyme can cleave the target.

[0171] Ribozymes can be introduced into cells as part of a DNAconstruct. Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease serine-threonine protein kinaseexpression. Alternatively, if it is desired that the cells stably retainthe DNA construct, the construct can be supplied on a plasmid andmaintained as a separate element or integrated into the genome of thecells, as is known in the art. A ribozyme-encoding DNA construct caninclude transcriptional regulatory elements, such as a promoter element,an enhancer or UAS element, and a transcriptional terminator signal, forcontrolling transcription of ribozymes in the cells.

[0172] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymescan be engineered so that ribozyme expression will occur in response tofactors which induce expression of a target gene. Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of mRNA occurs only when both a ribozyme and a target geneare induced in the cells.

[0173] Differentially Expressed Genes

[0174] Described herein are methods for the identification of geneswhose products interact with human serine-threonine protein kinase. Suchgenes may represent genes which are differentially expressed indisorders including, but not limited to, cancer, CNS disorders,diabetes, and COPD. Further, such genes may represent genes which aredifferentially regulated in response to manipulations relevant to theprogression or treatment of such diseases. Additionally, such genes mayhave a temporally modulated expression, increased or decreased atdifferent stages of tissue or organism development. A differentiallyexpressed gene may also have its expression modulated under controlversus experimental conditions. In addition, the human serine-threonineprotein kinase gene or gene product may itself be tested fordifferential expression.

[0175] The degree to which expression differs in a normal versus adiseased state need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

[0176] Identification of Differentially Expressed Genes

[0177] To identify differentially expressed genes total RNA or,preferably, mRNA is isolated from tissues of interest. For example, RNAsamples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquewhich does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Ausubel et al.,ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. NewYork, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

[0178] Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes are identified by methodswell known to those of skill in the art. They include, for example,differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85,208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308,149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and,preferably, differential display (Liang & Pardee, Science 257, 967-71,1992; U.S. Pat. No. 5,262,311).

[0179] The differential expression information may itself suggestrelevant methods for the treatment of disorders involving the humanserine-threonine protein kinase. For example, treatment may include amodulation of expression of the differentially expressed genes and/orthe gene encoding the human serine-threonine protein kinase. Thedifferential expression information may indicate whether the expressionor activity of the differentially expressed gene or gene product or thehuman serine-threonine protein kinase gene or gene product areup-regulated or down-regulated.

[0180] Screening Methods

[0181] The invention provides assays for screening test compounds whichbind to or modulate the activity of a serine-threonine protein kinasepolypeptide or a serine-threonine protein kinase polynucleotide. A testcompound preferably binds to a serine-threonine protein kinasepolypeptide or polynucleotide. More preferably, a test compounddecreases or increases activity by at least about 10, preferably about50, more preferably about 75, 90, or 100% relative to the absence of thetest compound.

[0182] Test Compounds

[0183] Test compounds can be pharmacologic agents already known in theart or can be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

[0184] Methods for the synthesis of molecular libraries are well knownin the art (see, for example, DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91,11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho etal., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl.33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061;Gallop et al., J. Med. Chem. 37, 1233, 1994). Libraries of compounds canbe presented in solution (see, e.g., Houghten, BioTechniques 13,412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips(Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S.Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad Sci. U.S.A.89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390,1990; Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl.Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310,1991; and Ladner, U.S. Pat. No. 5,223,409).

[0185] High Throughput Screening

[0186] Test compounds can be screened for the ability to bind toserine-threonine protein kinase polypeptides or polynucleotides or toaffect serine-threonine protein kinase activity or serine-threonineprotein kinase gene expression using high throughput screening. Usinghigh throughput screening, many discrete compounds can be tested inparallel so that large numbers of test compounds can be quicklyscreened. The most widely established techniques utilize 96-wellmicrotiter plates. The wells of the microtiter plates typically requireassay volumes that range from 50 to 500 μl. In addition to the plates,many instruments, materials, pipettors, robotics, plate washers, andplate readers are commercially available to fit the 96-well format.

[0187] Alternatively, “free format assays,” or assays that have nophysical barrier between samples, can be used. For example, an assayusing pigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

[0188] Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light. Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

[0189] Yet another example is described by Salmon et al., MolecularDiversity 2, 57-63 (1996). In this example, combinatorial libraries werescreened for compounds that had cytotoxic effects on cancer cellsgrowing in agar.

[0190] Another high throughput screening method is described in Beutelet al., U.S. Pat. No. 5,976,813. In this method, test samples are placedin a porous matrix. One or more assay components are then placed within,on top of, or at the bottom of a matrix such as a gel, a plastic sheet,a filter, or other form of easily manipulated solid support. Whensamples are introduced to the porous matrix they diffuse sufficientlyslowly, such that the assays can be performed without the test samplesrunning together.

[0191] Binding Assays

[0192] For binding assays, the test compound is preferably a smallmolecule which binds to and occupies, for example, the active site ofthe serine-threonine protein kinase polypeptide, such that normalbiological activity is prevented. Examples of such small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules.

[0193] In binding assays, either the test compound or theserine-threonine protein kinase polypeptide can comprise a detectablelabel, such as a fluorescent, radioisotopic, chemiluminescent, orenzymatic label, such as horseradish peroxidase, alkaline phosphatase,or luciferase. Detection of a test compound which is bound to theserine-threonine protein kinase polypeptide can then be accomplished,for example, by direct counting of radioemmission, by scintillationcounting, or by determining conversion of an appropriate substrate to adetectable product.

[0194] Alternatively, binding of a test compound to a serine-threonineprotein kinase polypeptide can be determined without labeling either ofthe interactants. For example, a microphysiometer can be used to detectbinding of a test compound with a serine-threonine protein kinasepolypeptide. A microphysiometer (e.g., Cytosensor™) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a test compound and a serine-threonine proteinkinase polypeptide (McConnell et al., Science 257, 1906-1912, 1992).

[0195] Determining the ability of a test compound to bind to aserine-threonine protein kinase polypeptide also can be accomplishedusing a technology such as real-time Bimolecular Interaction Analysis(BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, andSzabo et al., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore™). Changes in theoptical phenomenon surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

[0196] In yet another aspect of the invention, a serine-threonineprotein kinase polypeptide can be used as a “bait protein” in atwo-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al., Cell 72, 223-232, 1993; Madura et al., J.Biol. Chem. 268, 12046-12054, 1993; Bartel et al., BioTechniques 14,920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and BrentW094/10300), to identify other proteins which bind to or interact withthe serine-threonine protein kinase polypeptide and modulate itsactivity.

[0197] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding aserine-threonine protein kinase polypeptide can be fused to apolynucleotide encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct a DNA sequence that encodesan unidentified protein (“prey” or “sample”) can be fused to apolynucleotide that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract in vivo to form an protein-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ), which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected, and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the DNA sequenceencoding the protein which interacts with the serine-threonine proteinkinase polypeptide.

[0198] It may be desirable to immobilize either the serine-threonineprotein kinase polypeptide (or polynucleotide) or the test compound tofacilitate separation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the serine-threonine protein kinase polypeptide (orpolynucleotide) or the test compound can be bound to a solid support.Suitable solid supports include, but are not limited to, glass orplastic slides, tissue culture plates, microtiter wells, tubes, siliconchips, or particles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach the polypeptide (or polynucleotide) or test compound to a solidsupport, including use of covalent and non-covalent linkages, passiveabsorption, or pairs of binding moieties attached respectively to thepolypeptide (or polynucleotide) or test compound and the solid support.Test compounds are preferably bound to the solid support in an array, sothat the location of individual test compounds can be tracked. Bindingof a test compound to a serine-threonine protein kinase polypeptide (orpolynucleotide) can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and microcentrifuge tubes.

[0199] In one embodiment, the serine-threonine protein kinasepolypeptide is a fusion protein comprising a domain that allows theserine-threonine protein kinase polypeptide to be bound to a solidsupport. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and thenon-adsorbed serine-threonine protein kinase polypeptide; the mixture isthen incubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents. Binding of the interactants can be determined eitherdirectly or indirectly, as described above. Alternatively, the complexescan be dissociated from the solid support before binding is determined.

[0200] Other techniques for immobilizing proteins or polynucleotides ona solid support also can be used in the screening assays of theinvention. For example, either a serine-threonine protein kinasepolypeptide (or polynucleotide) or a test compound can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedserine-threonine protein kinase polypeptides (or polynucleotides) ortest compounds can be prepared from biotin-NHS(N-hydroxysuccinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.) and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies which specifically bind to a serine-threonine protein kinasepolypeptide, polynucleotide, or a test compound, but which do notinterfere with a desired binding site, such as the active site of theserine-threonine protein kinase polypeptide, can be derivatized to thewells of the plate. Unbound target or protein can be trapped in thewells by antibody conjugation.

[0201] Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies which specifically bind tothe serine-threonine protein kinase polypeptide or test compound,enzyme-linked assays which rely on detecting an activity of theserine-threonine protein kinase polypeptide, and SDS gel electrophoresisunder non-reducing conditions.

[0202] Screening for test compounds which bind to a serine-threonineprotein kinase polypeptide or polynucleotide also can be carried out inan intact cell. Any cell which comprises a serine-threonine proteinkinase polypeptide or polynucleotide can be used in a cell-based assaysystem. A serine-threonine protein kinase polynucleotide can benaturally occurring in the cell or can be introduced using techniquessuch as those described above. Binding of the test compound to aserine-threonine protein kinase polypeptide or polynucleotide isdetermined as described above.

[0203] Enzyme Assays

[0204] Test compounds can be tested for the ability to increase ordecrease the kinase activity of a human serine-threonine protein kinasepolypeptide. Kinase activity can be measured, for example, as describedin Example 4.

[0205] Enzyme assays can be carried out after contacting either apurified serine-threonine protein kinase polypeptide, a cell membranepreparation, or an intact cell with a test compound. A test compoundwhich decreases a kinase activity of a serine-threonine protein kinasepolypeptide by at least about 10, preferably about 50, more preferablyabout 75, 90, or 100% is identified as a potential therapeutic agent fordecreasing serine-threonine protein kinase activity. A test compoundwhich increases a kinase activity of a human serine-threonine proteinkinase polypeptide by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% is identified as a potentialtherapeutic agent for increasing human serine-threonine protein kinaseactivity.

[0206] Gene Expression

[0207] In another embodiment, test compounds which increase or decreaseserine-threonine protein kinase gene expression are identified. Aserine-threonine protein kinase polynucleotide is contacted with a testcompound, and the expression of an RNA or polypeptide product of theserine-threonine protein kinase polynucleotide is determined. The levelof expression of appropriate mRNA or polypeptide in the presence of thetest compound is compared to the level of expression of mRNA orpolypeptide in the absence of the test compound. The test compound canthen be identified as a modulator of expression based on thiscomparison. For example, when expression of mRNA or polypeptide isgreater in the presence of the test compound than in its absence, thetest compound is identified as a stimulator or enhancer of the mRNA orpolypeptide expression. Alternatively, when expression of the mRNA orpolypeptide is less in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor of the mRNA orpolypeptide expression.

[0208] The level of serine-threonine protein kinase mRNA or polypeptideexpression in the cells can be determined by methods well known in theart for detecting mRNA or polypeptide. Either qualitative orquantitative methods can be used. The presence of polypeptide productsof a serine-threonine protein kinase polynucleotide can be determined,for example, using a variety of techniques known in the art, includingimmunochemical methods such as radioimmunoassay, Western blotting, andimmunohistochemistry. Alternatively, polypeptide synthesis can bedetermined in vivo, in a cell culture, or in an in vitro translationsystem by detecting incorporation of labeled amino acids into aserine-threonine protein kinase polypeptide.

[0209] Such screening can be carried out either in a cell-free assaysystem or in an intact cell. Any cell which expresses a serine-threonineprotein kinase polynucleotide can be used in a cell-based assay system.The serine-threonine protein kinase polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Either a primary culture or an established cellline, such as CHO or human embryonic kidney 293 cells, can be used.

[0210] Pharmaceutical Compositions

[0211] The invention also provides pharmaceutical compositions which canbe administered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise, for example,a serine-threonine protein kinase polypeptide, serine-threonine proteinkinase polynucleotide, ribozymes or antisense oligonucleotides,antibodies which specifically bind to a serine-threonine protein kinasepolypeptide, or mimetics, activators, inhibitors, or inhibitors of aserine-threonine protein kinase polypeptide activity. The compositionscan be administered alone or in combination with at least one otheragent, such as stabilizing compound, which can be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions can be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

[0212] In addition to the active ingredients, these pharmaceuticalcompositions can contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0213] Pharmaceutical preparations for oral use can be obtained throughcombination of

[0214] active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers, such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0215] Dragee cores can be used in conjunction with suitable coatings,such as concentrated sugar solutions, which also can contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0216] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0217] Pharmaceutical formulations suitable for parenteraladministration can be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions can contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds can beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers also can be used for delivery.Optionally, the suspension also can contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. For topical or nasaladministration, penetrants appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0218] The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

[0219] Further details on techniques for formulation and administrationcan be found in the latest edition of REMINGTON'S PHARMACEUTICALSCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labeled for treatment of an indicated condition. Suchlabeling would include amount, frequency, and method of administration.

[0220] Therapeutic Indications and Methods

[0221] The human serine-threonine kinase disclosed herein is likely tobe useful for the same purposes as previously identifiedserine-threonine kinases. For example, transforming growth factor typebeta (TGF-β) regulates the proliferation and differentiation of avariety of cell types binding to and activating cell surface receptorswhich possess serine/threonine kinase activity. Atfi et al. (Proc. Natl.Acad. Sci. U.S.A. 92, 12110-04, 1995) have shown that TGF-β activates a78-kDa protein (p78) serine-threonine kinase; the p78 kinase wasactivated only in cells for which TGF-β acts as a growth inhibitoryfactor. The human serine-threonine kinase disclosed herein also may beinvolved in such signaling. Thus, regulation of its activity can be usedto treat disorders in which such signaling is defective.

[0222] Cancer. Cancer is a disease fundamentally caused by oncogeniccellular transformation. There are several hallmarks of transformedcells that distinguish them from their normal counterparts and underliethe pathophysiology of cancer. These include uncontrolled cellularproliferation, unresponsiveness to normal death-inducing signals(immortalization), increased cellular motility and invasiveness,increased ability to recruit blood supply through induction of new bloodvessel formation (angiogenesis), genetic instability, and dysregulatedgene expression. Various combinations of these aberrant physiologies,along with the acquisition of drug-resistance frequently lead to anintractable disease state in which organ failure and patient deathultimately ensue.

[0223] Most standard cancer therapies target cellular proliferation andrely on the differential proliferative capacities between transformedand normal cells for their efficacy. This approach is hindered by thefacts that several important normal cell types are also highlyproliferative and that cancer cells frequently become resistant to theseagents. Thus, the therapeutic indices for traditional anti-cancertherapies rarely exceed 2.0.

[0224] The advent of genomics-driven molecular target identification hasopened up the possibility of identifying new cancer-specific targets fortherapeutic intervention that will provide safer, more effectivetreatments for cancer patients. Thus, newly discovered tumor-associatedgenes and their products can be tested for their role(s) in disease andused as tools to discover and develop innovative therapies. Genesplaying important roles in any of the physiological processes outlinedabove can be characterized as cancer targets.

[0225] Genes or gene fragments identified through genomics can readilybe expressed in one or more heterologous expression systems to producefunctional recombinant proteins. These proteins are characterized invitro for their biochemical properties and then used as tools inhigh-throughput molecular screening programs to identify chemicalmodulators of their biochemical activities. Agonists and/or antagonistsof target protein activity can be identified in this manner andsubsequently tested in cellular and in vivo disease models foranti-cancer activity. Optimization of lead compounds with iterativetesting in biological models and detailed pharmacokinetic andtoxicological analyses form the basis for drug development andsubsequent testing in humans.

[0226] Diabetes. Diabetes mellitus is a common metabolic disordercharacterized by an abnormal elevation in blood glucose, alterations inlipids and abnormalities (complications) in the cardiovascular system,eye, kidney and nervous system. Diabetes is divided into two separatediseases: type 1 diabetes (juvenile onset), which results from a loss ofcells which make and secrete insulin, and type 2 diabetes (adult onset),which is caused by a defect in insulin secretion and a defect in insulinaction.

[0227] Type 1 diabetes is initiated by an autoimmune reaction thatattacks the insulin secreting cells (beta cells) in the pancreaticislets. Agents that prevent this reaction from occurring or that stopthe reaction before destruction of the beta cells has been accomplishedare potential therapies for this disease. Other agents that induce betacell proliferation and regeneration also are potential therapies.

[0228] Type II diabetes is the most common of the two diabeticconditions (6% of the population). The defect in insulin secretion is animportant cause of the diabetic condition and results from an inabilityof the beta cell to properly detect and respond to rises in bloodglucose levels with insulin release. Therapies that increase theresponse by the beta cell to glucose would offer an important newtreatment for this disease.

[0229] The defect in insulin action in Type II diabetic subjects isanother target for therapeutic intervention. Agents that increase theactivity of the insulin receptor in muscle, liver, and fat will cause adecrease in blood glucose and a normalization of plasma lipids. Thereceptor activity can be increased by agents that directly stimulate thereceptor or that increase the intracellular signals from the receptor.Other therapies can directly activate the cellular end process, i.e.glucose transport or various enzyme systems, to generate an insulin-likeeffect and therefore a produce beneficial outcome. Because overweightsubjects have a greater susceptibility to Type II diabetes, any agentthat reduces body weight is a possible therapy.

[0230] Both Type I and Type diabetes can be treated with agents thatmimic insulin action or that treat diabetic complications by reducingblood glucose levels. Likewise, agents that reduces new blood vesselgrowth can be used to treat the eye complications that develop in bothdiseases.

[0231] CNS disorders. CNS disorders which may be treated include braininjuries, cerebrovascular diseases and their consequences, Parkinson'sdisease, corticobasal degeneration, motor neuron disease, dementia,including ALS, multiple sclerosis, traumatic brain injury, stroke,post-stroke, post-traumatic brain injury, and small-vesselcerebrovascular disease. Dementias, such as Alzheimer's disease,vascular dementia, dementia with Lewy bodies, frontotemporal dementiaand Parkinsonism linked to chromosome 17, frontotemporal dementias,including Pick's disease, progressive nuclear palsy, corticobasaldegeneration, Huntington's disease, thalamic degeneration,Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia,and Korsakoff's psychosis also can be treated. Similarly, it may bepossible to treat cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders,attention deficit hyperactivity disorders, and memory disturbances inchildren with learning disabilities, by regulating the activity of humanserine/threonine protein kinase.

[0232] Pain that is associated with CNS disorders also can be treated byregulating the activity of human serine/threonine protein kinase. Painwhich can be treated includes that associated with central nervoussystem disorders, such as multiple sclerosis, spinal cord injury,sciatica, failed back surgery syndrome, traumatic brain injury,epilepsy, Parkinson's disease, post-stroke, and vascular lesions in thebrain and spinal cord (e.g., infarct, hemorrhage, vascularmalformation). Non-central neuropathic pain includes that associatedwith post mastectomy pain, reflex sympathetic dystrophy (RSD),trigeminal neuralgiaradioculopathy, post-surgical pain, HTV/AIDS relatedpain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neuralgias, and post-herpeticneuralgia. Pain associated with cancer and cancer treatment also can betreated, as can headache pain (for example, migraine with aura, migrainewithout aura, and other migraine disorders), episodic and chronictension-type headache, tension-type like headache, cluster headache, andchronic paroxysmal hemicrania.

[0233] COPD. Chronic obstructive pulmonary (or airways) disease (COPD)is a condition defined physiologically as airflow obstruction thatgenerally results from a mixture of emphysema and peripheral airwayobstruction due to chronic bronchitis (Senior & Shapiro, PulmonaryDiseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp.659-681, 1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema ischaracterized by destruction of alveolar walls leading to abnormalenlargement of the air spaces of the lung. Chronic bronchitis is definedclinically as the presence of chronic productive cough for three monthsin each of two successive years. In COPD, airflow obstruction is usuallyprogressive and is only partially reversible. By far the most importantrisk factor for development of COPD is cigarette smoking, although thedisease does occur in non-smokers.

[0234] Chronic inflammation of the airways is a key pathological featureof COPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages which are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

[0235] COPD is characterized by damage to the lung extracellular matrixand emphysema can be viewed as the pathologic process that affects thelung parenchyma. This process eventually leads to the destruction of theairway walls resulting in permanent airspace enlargement (Senior andShapiro, in PULMONARY DISEASES AND DISORDERS, 3^(rd) ed., New York,McGraw-Hill, 1998, pp. 659-681, 1998). The observation that inheriteddeficiency of al-antitrypsin (al-AT), the primary inhibitor ofneutrophil elastase, predisposes individuals to early onset emphysema,and that intrapulmonary instillation of elastolytic enzymes inexperimental animals causes emphysema, led to the elastase:antielastasehypothesis for the pathogenesis of emphysema (Eriksson, Acta Med. Scand.177(Suppl.), 432, 1965, Gross, J. Occup. Med. 6, 481-84, 1964). This inturn led to the concept that destruction of elastin in the lungparenchyma is the basis of the development of emphysema.

[0236] A broad range of immune and inflammatory cells includingneutrophils, macrophages, T lymphocytes and eosinophils containproteolytic enzymes that could contribute to the destruction of lungextracellular matrix (Shapiro, 1999). In addition, a number of differentclasses of proteases have been identified that have the potential tocontribute to lung matrix destruction. These include serine proteases,matrix metalloproteinases and cysteine proteases. Of these classes ofenzymes, a number can hydrolyze elastin and have been shown to beelevated in COPD patients (neutrophil elastase, MMP-2, 9, 12) (Culpittet al., Am. J. Respir. Crit. Care Med. 160, 1635-39, 1999, Shapiro, Am.J. Crit. Care Med. 160 (5), S29- S32,1999).

[0237] It is expected that in the future novel members of the existingclasses of proteases and new classes of proteases will be identifiedthat play a significant role in the damage of the extracellular lungmatrix including elastin proteolysis. Novel protease targets thereforeremain very attractive therapeutic targets.

[0238] This invention further pertains to the use of novel agentsidentified by the screening assays described above. Accordingly, it iswithin the scope of this invention to use a test compound identified asdescribed herein in an appropriate animal model. For example, an agentidentified as described herein (e.g., a modulating agent, an antisensenucleic acid molecule, a specific antibody, ribozyme, or aserine-threonine protein kinase polypeptide binding molecule) can beused in an animal model to determine the efficacy, toxicity, or sideeffects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal model todetermine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

[0239] A reagent which affects serine-threonine protein kinase activitycan be administered to a human cell, either in vitro or in vivo, toreduce serine-threonine protein kinase activity. The reagent preferablybinds to an expression product of a human serine-threonine proteinkinase gene. If the expression product is a protein, the reagent ispreferably an antibody. For treatment of human cells ex vivo, anantibody can be added to a preparation of stem cells which have beenremoved from the body. The cells can then be replaced in the same oranother human body, with or without clonal propagation, as is known inthe art.

[0240] In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung, liver, spleen, heart brain, lymph nodes, and skin.

[0241] A liposome useful in the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of thetargeted cell to deliver its contents to the cell. Preferably, thetransfection efficiency of a liposome is about 0.5 μg of DNA per 16nmole of liposome delivered to about 10⁶ cells, more preferably about1.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells, andeven more preferably about 2.0 μg of DNA per 16 nmol of liposomedelivered to about 10⁶ cells. Preferably, a liposome is between about100 and 500 nm, more preferably between about 150 and 450 nm, and evenmore preferably between about 200 and 400 nm in diameter.

[0242] Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to aparticular cell type, such as a cell-specific ligand exposed on theouter surface of the liposome.

[0243] Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods which arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

[0244] In another embodiment, antibodies can be delivered to specifictissues in vivo using receptor-mediated targeted delivery.Receptor-mediated DNA delivery techniques are taught in, for example,Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al.,GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu etal., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad.Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42(1991).

[0245] Determination of a Therapeutically Effective Dose

[0246] The determination of a therapeutically effective dose is wellwithin the capability of those skilled in the art. A therapeuticallyeffective dose refers to that amount of active ingredient whichincreases or decreases serine-threonine protein kinase activity relativeto the serine-threonine protein kinase activity which occurs in theabsence of the therapeutically effective dose.

[0247] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays or in animal models,usually mice, rabbits, dogs, or pigs. The animal model also can be usedto determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

[0248] Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population), can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀.

[0249] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0250] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

[0251] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0252] If the reagent is a single-chain antibody, polynucleotidesencoding the antibody can be constructed and introduced into a celleither ex vivo or in vivo using well-established techniques including,but not limited to, transferrin-polycation-mediated DNA transfer,transfection with naked or encapsulated nucleic acids, liposome-mediatedcellular fusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, “gene gun,” andDEAE- or calcium phosphate-mediated transfection.

[0253] Effective in vivo dosages of an antibody are in the range ofabout 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μg/kg of patient body weight. For administration of polynucleotidesencoding single-chain antibodies, effective in vivo dosages are in therange of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μgto about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100μg of DNA.

[0254] If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides which expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

[0255] Preferably, a reagent reduces expression of a serine-threonineprotein kinase gene or the activity of a serine-threonine protein kinasepolypeptide by at least about 10, preferably about 50, more preferablyabout 75, 90, or 100% relative to the absence of the reagent. Theeffectiveness of the mechanism chosen to decrease the level ofexpression of a serine-threonine protein kinase gene or the activity ofa serine-threonine protein kinase polypeptide can be assessed usingmethods well known in the art, such as hybridization of nucleotideprobes to serine-threonine protein kinase-specific mRNA, quantitativeRT-PCR, immunologic detection of a serine-threonine protein kinasepolypeptide, or measurement of serine-threonine protein kinase activity.

[0256] In any of the embodiments described above, any of thepharmaceutical compositions of the invention can be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy can be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents can actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

[0257] Any of the therapeutic methods described above can be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0258] Diagnostic Methods

[0259] Human serine-threonine protein kinase also can be used indiagnostic assays for detecting diseases and abnormalities orsusceptibility to diseases and abnormalities related to the presence ofmutations in the nucleic acid sequences which encode the enzyme. Forexample, differences can be determined between the cDNA or genomicsequence encoding serine-threonine protein kinase in individualsafflicted with a disease and in normal individuals. If a mutation isobserved in some or all of the afflicted individuals but not in normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

[0260] Sequence differences between a reference gene and a gene havingmutations can be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments can be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer can beused with a double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures using radiolabeled nucleotides orby automatic sequencing procedures using fluorescent tags.

[0261] Genetic testing based on DNA sequence differences can be carriedout by detection of alteration in electrophoretic mobility of DNAfragments in gels with or without denaturing agents. Small sequencedeletions and insertions can be visualized, for example, by highresolution gel electrophoresis. DNA fragments of different sequences canbe distinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985).Sequence changes at specific locations can also be revealed by nucleaseprotection assays, such as RNase and S 1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85,4397-4401, 1985). Thus, the detection of a specific DNA sequence can beperformed by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes andSouthern blotting of genomic DNA. In addition to direct methods such asgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

[0262] Altered levels of a serine-threonine protein kinase also can bedetected in various tissues. Assays used to detect levels of thereceptor polypeptides in a body sample, such as blood or a tissuebiopsy, derived from a host are well known to those of skill in the artand include radioimmunoassays, competitive binding assays, Western blotanalysis, and ELISA assays.

[0263] All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLE 1

[0264] Detection of Serine-Threonine Protein Kinase Activity

[0265] For high level expression of a FLAG-tagged serine-threonineprotein kinase polypeptide, COS-1 cells are transfected with theexpression vector serine-threonine protein kinase polypeptide(expressing the DNA-sequence of SEQ ID NO: 1 or 16) using the calciumphosphate method. After 5 h, the cells are infected with recombinantvaccinia virus vTF7-3 (10 plaque-forming units/cell). The cells areharvested 20 h after infection and lysed in 50 mM Tris, pH 7.5, 5 mMMgCl2, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 1 mMphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin. Serine-threonineprotein kinase polypeptide is immunoprecipitated from the lysate usinganti-FLAG antibodies. In vitro kinase assay and phosphoamino acidanalysis are performed in a volume of 40 μl with immunoprecipitatedFLAG-serine-threonine protein kinase polypeptide in 50 mM Tris-HCl, pH8.0, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol. The reaction isstarted by the addition of 4 μl of 1 mM ATP supplemented with 5 μCi of(−32P)ATP and incubated for 30 min at 37° C. Afterward, the samples aresubjected to SDS-PAGE and phosphorylated proteins are detected byautoradiography. Histone type III-S, casein, bovine serum albumin, ormyelin basic proteins are used as substrates. It is shown that thepolypeptide with the amino acid sequence of SEQ ID NO.: 2 and 17respectively have serine-threonine protein kinase activity.

EXAMPLE 2

[0266] Expression of Recombinant Human Serine-Threonine Protein Kinase

[0267] The Pichia pastoris expression vector pPICZB (Invitrogen, SanDiego, Calif.) is used to produce large quantities of recombinant humanserine-threonine protein kinase polypeptides in yeast. Theserine-threonine protein kinase-encoding DNA sequence is derived fromSEQ ID NO:1 or SEQ ID NO:16. Before insertion into vector pPICZB, theDNA sequence is modified by well known methods in such a way that itcontains at its 5′-end an initiation codon and at its 3′-end anenterokinase cleavage site, a His6 reporter tag and a termination codon.Moreover, at both termini recognition sequences for restrictionendonucleases are added and after digestion of the multiple cloning siteof pPICZ B with the corresponding restriction enzymes the modified DNAsequence is ligated into pPICZB. This expression vector is designed forinducible expression in Pichia pastoris, driven by a yeast promoter. Theresulting pPICZ/md-His6 vector is used to transform the yeast.

[0268] The yeast is cultivated under usual conditions in 5 liter shakeflasks and the recombinantly produced protein isolated from the cultureby affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.The bound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humanserine-threonine protein kinase polypeptide is obtained.

EXAMPLE 3

[0269] Identification of Test Compounds That Bind to Serine-ThreonineProtein Kinase Polypeptides

[0270] Purified serine-threonine protein kinase polypeptides comprisinga glutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Human serine-threonine protein kinasepolypeptides comprise the amino acid sequence shown in SEQ ID NO:2 orSEQ ID NO:17. The test compounds comprise a fluorescent tag. The samplesare incubated for 5 minutes to one hour. Control samples are incubatedin the absence of a test compound.

[0271] The buffer solution containing the test compounds is washed fromthe wells. Binding of a test compound to a serine-threonine proteinkinase polypeptide is detected by fluorescence measurements of thecontents of the wells. A test compound which increases the fluorescencein a well by at least 15% relative to fluorescence of a well in which atest compound is not incubated is identified as a compound which bindsto a serine-threonine protein kinase polypeptide.

EXAMPLE 4

[0272] Identification of a Test Compound Which DecreasesSerine-Threonine Protein Kinase Gene Expression

[0273] A test compound is administered to a culture of human cellstransfected with a serine-threonine protein kinase expression constructand incubated at 37° C. for 10 to 45 minutes. A culture of the same typeof cells which have not been transfected is incubated for the same timewithout the test compound to provide a negative control.

[0274] RNA is isolated from the two cultures as described in Chirgwin etal., Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20to 30 μg total RNA and hybridized with a ³²P-labeled serine-threonineprotein kinase-specific probe at 65° C. in Express-hyb (CLONTECH). Theprobe comprises at least 11 contiguous nucleotides selected from thecomplement of SEQ ID NO:1 or SEQ ID NO:16. A test compound whichdecreases the serine-threonine protein kinase-specific signal relativeto the signal obtained in the absence of the test compound is identifiedas an inhibitor of serine-threonine protein kinase gene expression.

EXAMPLE 5

[0275] Identification of a Test Compound Which Decreases HumanSerine-Threonine Protein Kinase Activity

[0276] Cellular extracts from the human colon cancer cell line HCT116are contacted with test compounds from a small molecule library andassayed for human serine-threonine protein kinase activity. Controlextracts, in the absence of a test compound, also are assayed. Kinaseactivity can be measured, for example, as taught in Trost et al., J.Biol. Chem. 275, 7373-77, 2000; Hayashi et al., Biochem. Biophys. Res.Commun. 264, 449-56, 1999; Masure et al., Eur. J Biochem. 265, 353-60,1999; and Mukhopadhyay et al., J. Bacteriol. 181, 6615-22, 1999. A testcompound which decreases serine-threonine protein kinase activity of theextract relative to the control extract by at least 20% is identified asa serine-threonine protein kinase inhibitor.

EXAMPLE 6

[0277] Tissue-Specific Expression of Serine/Threonine Protein Kinase

[0278] The qualitative expression pattern of serine/threonine proteinkinase in various tissues is determined by ReverseTranscription-Polymerase Chain Reaction (RT-PCR).

[0279] To demonstrate that serine/threonine protein kinase is involvedin cancer, expression is determined in the following tissues: adrenalgland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver,heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,stomach, testis, thymus, thyroid, trachea, uterus, and peripheral bloodlymphocytes. Expression in the following cancer cell lines also isdetermined: DU145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205(colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon),MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435(breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignantand normal tissue from the same patient also are tested.

[0280] To demonstrate that serine/threonine protein kinase is involvedin the disease process of COPD, the initial expression panel consists ofRNA samples from respiratory tissues and inflammatory cells relevant toCOPD: lung (adult and fetal), trachea, freshly isolated alveolar type IIcells, cultured human bronchial epithelial cells, cultured small airwayepithelial cells, cultured bronchial sooth muscle cells, cultured H441cells (Clara-like), freshly isolated neutrophils and monocytes, andcultured monocytes (macrophage-like). Body map profiling also is carriedout, using total RNA panels purchased from Clontech. The tissues areadrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung,mammary gland, pancreas, prostate, salivary gland, skeletal muscle,small intestine, spleen, stomach, testis, thymus, trachea, thyroid, anduterus.

[0281] To demonstrate that serine/threonine protein kinase is involvedin the disease process of obesity, expression is determined in thefollowing tissues: subcutaneous adipose tissue, mesenteric adiposetissue, adrenal gland, bone marrow, brain (cerebellum, spinal cord,cerebral cortex, caudate, medulla, substantia nigra, and putamen),colon, fetal brain, heart, kidney, liver, lung, mammary gland, pancreas,placenta, prostate, salivary gland, skeletal muscle small intestine,spleen, stomach, testes, thymus, thyroid trachea, and uterus.Neuroblastoma cell lines SK-Nr-Be (2), Hr, Sk-N-As, HTB-10, IMR-32,SNSY-5Y, T3, SK-N-D2, D283, DAOY, CHP-2, U87MG, BE(2)C, T986, KANTS,MO59K, CHP234, C6 (rat), SK-N-F1, SK-PU-DW, PFSK1, BE(2)M17, and MCIXCalso are tested for serine/threonine protein kinase expression. As afinal step, the expression of serine/threonine protein kinase in cellsderived from normal individuals with the expression of cells derivedfrom obese individuals is compared.

[0282] To demonstrate that serine/threonine protein kinase is involvedin the disease process of diabetes, the following whole body panel isscreened to show predominant or relatively high expression: subcutaneousand mesenteric adipose tissue, adrenal gland, bone marrow, brain, colon,fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland,pancreas, placenta, prostate, salivary gland, skeletal muscle, smallintestine, spleen, stomach, testis, thymus, thyroid, trachea, anduterus. Human islet cells and an islet cell library also are tested. Asa final step, the expression of serine/threonine protein kinase in cellsderived from normal individuals with the expression of cells derivedfrom diabetic individuals is compared.

[0283] To demonstrate that serine/threonine protein kinase is involvedin CNS disorders, the following tissues are screened: fetal and adultbrain, muscle, heart, lung, kidney, liver, thymus, testis, colon,placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver,cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex,amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinalcord.

[0284] Quantitative expression profiling. Quantitative expressionprofiling is performed by the form of quantitative PCR analysis called“kinetic analysis” firstly described in Higuchi et al., BioTechnology10, 413-17, 1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993.The principle is that at any given cycle within the exponential phase ofPCR, the amount of product is proportional to the initial number oftemplate copies.

[0285] If the amplification is performed in the presence of aninternally quenched fluorescent oligonucleotide (TaqMan probe)complementary to the target sequence, the probe is cleaved by the 5′-3′endonuclease activity of Taq DNA polymerase and a fluorescent dyereleased in the medium (Holland et al., Proc. Natl. Acad. Sci. U.S.A.88, 7276-80, 1991). Because the fluorescence emission will increase indirect proportion to the amount of the specific amplified product, theexponential growth phase of PCR product can be detected and used todetermine the initial template concentration (Heid et al., Genome Res.6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).

[0286] The amplification of an endogenous control can be performed tostandardize the amount of sample RNA added to a reaction. In this kindof experiment, the control of choice is the 18S ribosomal RNA. Becausereporter dyes with differing emission spectra are available, the targetand the endogenous control can be independently quantified in the sametube if probes labeled with different dyes are used.

[0287] All “real time PCR” measurements of fluorescence are made in theABI Prism 7700.

[0288] RNA extraction and cDNA preparation. Total RNA from the tissueslisted above are used for expression quantification. RNAs labeled “fromautopsy” were extracted from autoptic tissues with the TRIzol reagent(Life Technologies, MD) according to the manufacturer's protocol.

[0289] Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C. in the following reaction mix: 0.2 U/μl RNase-free DNase I (RocheDiagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems,CA); 10 mM Tris-HCl pH 7.9; 10 mM MgCl₂; 50 mM NaCl; and 1 mM DTT.

[0290] After incubation, RNA is extracted once with 1 volume ofphenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform,and precipitated with {fraction (1/10)} volume of 3 M NaAcetate, pH5.2,and 2 volumes of ethanol.

[0291] Fifty μg of each RNA from the autoptic tissues are DNase treatedwith the DNA-free kit purchased from Ambion (Ambion, Tex.). Afterresuspension and spectrophotometric quantification, each sample isreverse transcribed with the TaqMan Reverse Transcription Reagents (PEApplied Biosystems, CA) according to the manufacturer's protocol. Thefinal concentration of RNA in the reaction mix is 200 ng/μL. Reversetranscription is carried out with 2.5 μM of random hexamer primers.

[0292] TaqMan quantitative analysis. Specific primers and probe aredesigned according to the recommendations of PE Applied Biosystems; theprobe can be labeled at the 5′ end FAM (6-carboxy-fluorescein) and atthe 3′ end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantificationexperiments are performed on 10 ng of reverse transcribed RNA from eachsample. Each determination is done in triplicate.

[0293] Total cDNA content is normalized with the simultaneousquantification (multiplex PCR) of the 18S ribosomal RNA using thePre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE AppliedBiosystems, CA).

[0294] The assay reaction mix is as follows: 1X final TaqMan UniversalPCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); 1X PDARcontrol—18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverseprimer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

[0295] Each of the following steps are carried out once: pre PCR, 2minutes at 50° C., and 10 minutes at 95° C. The following steps arecarried out 40 times: denaturation, 15 seconds at 95° C.,annealing/extension, 1 minute at 60° C.

[0296] The experiment is performed on an ABI Prism 7700 SequenceDetector (PE Applied Biosystems, CA). At the end of the run,fluorescence data acquired during PCR are processed as described in theABI Prism 7700 user's manual in order to achieve better backgroundsubtraction as well as signal linearity with the starting targetquantity.

EXAMPLE 7

[0297] In Vivo Testing of Compounds/Target Validation

[0298] 1. Pain:

[0299] Acute Pain

[0300] Acute pain is measured on a hot plate mainly in rats. Twovariants of hot plate testing are used: In the classical variant animalsare put on a hot surface (52 to 56° C.) and the latency time is measureduntil the animals show nocifensive behavior, such as stepping or footlicking. The other variant is an increasing temperature hot plate wherethe experimental animals are put on a surface of neutral temperature.Subsequently this surface is slowly but constantly heated until theanimals begin to lick a hind paw. The temperature which is reached whenhind paw licking begins is a measure for pain threshold.

[0301] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to pain testing.

[0302] Persistent Pain

[0303] Persistent pain is measured with the formalin or capsaicin test,mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicinis injected into one hind paw of the experimental animal. After formalinor capsaicin application the animals show nocifensive reactions likeflinching, licking and biting of the affected paw. The number ofnocifensive reactions within a time frame of up to 90 minutes is ameasure for intensity of pain.

[0304] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to formalin or capsaicin administration.

[0305] Neuropathic Pain

[0306] Neuropathic pain is induced by different variants of unilateralsciatic nerve injury mainly in rats. The operation is performed underanesthesia. The first variant of sciatic nerve injury is produced byplacing loosely constrictive ligatures around the common sciatic nerve.The second variant is the tight ligation of about the half of thediameter of the common sciatic nerve. In the next variant, a group ofmodels is used in which tight ligations or transections are made ofeither the L5 and L6 spinal nerves, or the L % spinal nerve only. Thefourth variant involves an axotomy of two of the three terminal branchesof the sciatic nerve (tibial and common peroneal nerves) leaving theremaining sural nerve intact whereas the last variant comprises theaxotomy of only the tibial branch leaving the sural and common nervesuninjured. Control animals are treated with a sham operation.

[0307] Postoperatively, the nerve injured animals develop a chronicmechanical allodynia, cold allodynioa, as well as a thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA; Electronic von Frey System,Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is measured bymeans of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy), or by means of a cold plate of 5 to 10° C. where the nocifensivereactions of the affected hind paw are counted as a measure of painintensity. A further test for cold induced pain is the counting ofnocifensive reactions, or duration of nocifensive responses afterplantar administration of acetone to the affected hind limb. Chronicpain in general is assessed by registering the circadanian rhythms inactivity (Surjo and Arndt, Universität zu Köln, Cologne, Germany), andby scoring differences in gait (foot print patterns; FOOTPRINTS program,Klapdor et al., 1997. A low cost method to analyze footprint patterns.J. Neurosci. Methods 75, 49-54).

[0308] Compounds are tested against sham operated and vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

[0309] Inflammatory Pain

[0310] Inflammatory pain is induced mainly in rats by injection of 0.75mg carrageenan or complete Freund's adjuvant into one hind paw. Theanimals develop an edema with mechanical allodynia as well as thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measuredby means of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).For edema measurement two methods are being used. In the first method,the animals are sacrificed and the affected hindpaws sectioned andweighed. The second method comprises differences in paw volume bymeasuring water displacement in a plethysmometer (Ugo Basile, Comerio,Italy).

[0311] Compounds are tested against uninflamed as well as vehicletreated control groups. Substance application is performed at differenttime points via different application routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0312] Diabetic Neuropathic Pain

[0313] Rats treated with a single intraperitoneal injection of 50 to 80mg/kg streptozotocin develop a profound hyperglycemia and mechanicalallodynia within 1 to 3 weeks. Mechanical allodynia is measured by meansof a pressure transducer (electronic von Frey Anesthesiometer, IITCInc.-Life Science Instruments, Woodland Hills, SA, USA).

[0314] Compounds are tested against diabetic and non-diabetic vehicletreated control groups. Substance application is performed at differenttime points via different application routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0315] 2. Parkinson's Disease

[0316] 6-Hydroxydopamine (6-OH-DA) Lesion

[0317] Degeneration of the dopaminergic nigrostriatal andstriatopallidal pathways is the central pathological event inParkinson's disease. This disorder has been mimicked experimentally inrats using single/sequential unilateral stereotaxic injections of6-OH-DA into the medium forebrain bundle (MFB).

[0318] Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 gat the beginning of the experiment, are used. The rats are maintained ina temperature- and humidity-controlled environment under a 12 hlight/dark cycle with free access to food and water when not inexperimental sessions. The following in vivo protocols are approved bythe governmental authorities. All efforts are made to minimize animalsuffering, to reduce the number of animals used, and to utilizealternatives to in vivo techniques.

[0319] Animals are administered pargyline on the day of surgery (Sigma,St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of6-OHDA by monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kgi.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals.Thirty minutes later the rats are anesthetized with sodium pentobarbital(50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DAnigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μgof 6-OHDA HBr (Sigma) are injected into the left medial fore-brainbundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mmventral to Bregma and the skull surface). The needle is left in place anadditional 5 min to allow diffusion to occur.

[0320] Stepping Test

[0321] Forelimb akinesia is assessed three weeks following lesionplacement using a modified stepping test protocol. In brief, the animalsare held by the experimenter with one hand fixing the hindlimbs andslightly raising the hind part above the surface. One paw is touchingthe table, and is then moved slowly sideways (5 s for 1 m), first in theforehand and then in the backhand direction. The number of adjustingsteps is counted for both paws in the backhand and forehand direction ofmovement. The sequence of testing is right paw forehand and backhandadjusting stepping, followed by left paw forehand and backhanddirections. The test is repeated three times on three consecutive days,after an initial training period of three days prior to the firsttesting. Forehand adjusted stepping reveals no consistent differencesbetween lesioned and healthy control animals. Analysis is thereforerestricted to backhand adjusted stepping.

[0322] Balance Test

[0323] Balance adjustments following postural challenge are alsomeasured during the stepping test sessions. The rats are held in thesame position as described in the stepping test and, instead of beingmoved sideways, tilted by the experimenter towards the side of the pawtouching the table. This maneuver results in loss of balance and theability of the rats to regain balance by forelimb movements is scored ona scale ranging from 0 to 3. Score 0 is given for a normal forelimbplacement. When the forelimb movement is delayed but recovery ofpostural balance detected, score 1 is given. Score 2 represents a clear,yet insufficient, forelimb reaction, as evidenced by muscle contraction,but lack of success in recovering balance, and score 3 is given for noreaction of movement. The test is repeated three times a day on eachside for three consecutive days after an initial training period ofthree days prior to the first testing.

[0324] Staircase Test (Paw Reaching)

[0325] A modified version of the staircase test is used for evaluationof paw reaching behavior three weeks following primary and secondarylesion placement. Plexiglass test boxes with a central platform and aremovable staircase on each side are used. The apparatus is designedsuch that only the paw on the same side at each staircase can be used,thus providing a measure of independent forelimb use. For each test theanimals are left in the test boxes for 15 min. The double staircase isfilled with 7×3 chow pellets (Precision food pellets, formula: P,purified rodent diet, size 45 mg; Sandown Scientific) on each side.After each test the number of pellets eaten (successfully retrievedpellets) and the number of pellets taken (touched but dropped) for eachpaw and the success rate (pellets eaten/pellets taken) are countedseparately. After three days of food deprivation (12 g per animal perday) the animals are tested for 11 days. Full analysis is conducted onlyfor the last five days.

[0326] MPTP Treatment

[0327] The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP) causes degeneration of mesencephalic dopaminergic (DAergic)neurons in rodents, non-human primates, and humans and, in so doing,reproduces many of the symptoms of Parkinson's disease. MPTP leads to amarked decrease in the levels of dopamine and its metabolites, and inthe number of dopaminergic terminals in the striatum as well as severeloss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in thesubstantia nigra, pars compacta.

[0328] In order to obtain severe and long-lasting lesions, and to reducemortality, animals receive single injections of MPTP, and are thentested for severity of lesion 7-10 days later. Successive MPTPinjections are administered on days 1, 2 and 3. Animals receiveapplication of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily.All injections are intraperitoneal (i.p.) and the MPTP stock solution isfrozen between injections. Animals are decapitated on day 11.

[0329] Immunohistology

[0330] At the completion of behavioral experiments, all animals areanaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). Themice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min,followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brainsare removed and placed in 4% paraformaldehyde for 24 h at 4° C. Fordehydration they are then transferred to a 20% sucrose (Merck) solutionin 0.1 M PBS at 4° C. until they sink. The brains are frozen inmethylbutan at −20° C. for 2 min and stored at −70° C. Using a sledgemicrotome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from thegenu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm)and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored inassorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

[0331] A series of sections is processed for free-floating tyrosinehydroxylase (TH) immunohistochemistry. Following three rinses in 0.1 MPBS, endogenous peroxidase activity is quenched for 10 min in 0.3%H₂O₂±PBS. After rinsing in PBS, sections are preincubated in 10% normalbovine serum (Sigma) for 5 min as blocking agent and transferred toeither primary anti-rat TH rabbit antiserum (dilution 1:2000).

[0332] Following overnight incubation at room temperature, sections forTH immunoreactivity are rinsed in PBS (2×10 min) and incubated inbiotinylated anti-rabbit immunoglobulin G raised in goat (dilution1:200) (Vector) for 90 min, rinsed repeatedly and transferred toVectastain ABC (Vector) solution for 1 h. 3,.3′ -Diaminobenzidinetetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%H₂O₂, serves as chromogen in the subsequent visualization reaction.Sections are mounted on to gelatin-coated slides, left to dry overnight,counter-stained with hematoxylin dehydrated in ascending alcoholconcentrations and cleared in butylacetate. Coverslips are mounted onentellan.

[0333] Rotarod Test

[0334] We use a modification of the procedure described by Rozas andLabandeira-Garcia (1997), with a CR-1 Rotamex system (ColumbusInstruments, Columbus, Ohio) comprising an IBM-compatible personalcomputer, a CIO-24 data acquisition card, a control unit, and afour-lane rotarod unit. The rotarod unit consists of a rotating spindle(diameter 7.3 cm) and individual compartments for each mouse. The systemsoftware allows preprogramming of session protocols with varyingrotational speeds (0-80 rpm). Infrared beams are used to detect when amouse has fallen onto the base grid beneath the rotarod. The system logsthe fall as the end of the experiment for that mouse, and the total timeon the rotarod, as well as the time of the fall and all the set-upparameters, are recorded. The system also allows a weak current to bepassed through the base grid, to aid training.

[0335] 3. Dementia

[0336] The Object Recognition Task

[0337] The object recognition task has been designed to assess theeffects of experimental manipulations on the cognitive performance ofrodents. A rat is placed in an open field, in which two identicalobjects are present. The rats inspects both objects during the firsttrial of the object recognition task. In a second trial, after aretention interval of for example 24 hours, one of the two objects usedin the first trial, the ‘familiar’ object, and a novel object are placedin the open field. The inspection time at each of the objects isregistered. The basic measures in the OR task is the time spent by a ratexploring the two object the second trial. Good retention is reflectedby higher exploration times towards the novel than the ‘familiar’object.

[0338] Administration of the putative cognition enhancer prior to thefirst trial predominantly allows assessment of the effects onacquisition, and eventually on consolidation processes. Administrationof the testing compound after the first trial allows to assess theeffects on consolidation processes, whereas administration before thesecond trial allows to measure effects on retrieval processes.

[0339] The Passive Avoidance Task

[0340] The passive avoidance task assesses memory performance in ratsand mice. The inhibitory avoidance apparatus consists of atwo-compartment box with a light compartment and a dark compartment. Thetwo compartments are separated by a guillotine door that can be operatedby the experimenter. A threshold of 2 cm separates the two compartmentswhen the guillotine door is raised. When the door is open, theillumination in the dark compartment is about 2 lux. The light intensityis about 500 lux at the center of the floor of the light compartment.

[0341] Two habituation sessions, one shock session, and a retentionsession are given, separated by inter-session intervals of 24 hours. Inthe habituation sessions and the retention session the rat is allowed toexplore the apparatus for 300 sec. The rat is placed in the lightcompartment, facing the wall opposite to the guillotine door. After anaccommodation period of 15 sec. the guillotine door is opened so thatall parts of the apparatus can be visited freely. Rats normally avoidbrightly lit areas and will enter the dark compartment within a fewseconds.

[0342] In the shock session the guillotine door between the compartmentsis lowered as soon as the rat has entered the dark compartment with itsfour paws, and a scrambled 1 mA footshock is administered for 2 sec. Therat is removed from the apparatus and put back into its home cage. Theprocedure during the retention session is identical to that of thehabituation sessions.

[0343] The step-through latency, that is the first latency of enteringthe dark compartment (in sec.) during the retention session is an indexof the memory performance of the animal; the longer the latency to enterthe dark compartment, the better the retention is. A testing compound ingiven half an hour before the shock session, together with 1 mg*kg⁻¹scopolamine. Scopolamine impairs the memory performance during theretention session 24 hours later. If the test compound increases theenter latency compared with the scopolamine-treated controls, is likelyto possess cognition enhancing potential.

[0344] The Morris Water Escape Task

[0345] The Morris water escape task measures spatial orientationlearning in rodents. It is a test system that has extensively been usedto investigate the effects of putative therapeutic on the cognitivefunctions of rats and mice. The performance of an animal is assessed ina circular water tank with an escape platform that is submerged about 1cm below the surface of the water. The escape platform is not visiblefor an animal swimming in the water tank. Abundant extra-maze cues areprovided by the furniture in the room, including desks, computerequipment, a second water tank, the presence of the experimenter, and bya radio on a shelf that is playing softly.

[0346] The animals receive four trials during five daily acquisitionsessions. A trial is started by placing an animal into the pool, facingthe wall of the tank. Each of four starting positions in the quadrantsnorth, east, south, and west is used once in a series of four trials;their order is randomized. The escape platform is always in the sameposition. A trial is terminated as soon as the animal had climbs ontothe escape platform or when 90 seconds have elapsed, whichever eventoccurs first. The animal is allowed to stay on the platform for 30seconds. Then it is taken from the platform and the next trial isstarted. If an animal did not find the platform within 90 seconds it isput on the platform by the experimenter and is allowed to stay there for30 seconds. After the fourth trial of the fifth daily session, anadditional trial is given as a probe trial: the platform is removed, andthe time the animal spends in the four quadrants is measured for 30 or60 seconds. In the probe trial, all animals start from the same startposition, opposite to the quadrant where the escape platform had beenpositioned during acquisition.

[0347] Four different measures are taken to evaluate the performance ofan animal during acquisition training: escape latency, traveleddistance, distance to platform, and swimming speed. The followingmeasures are evaluated for the probe trial: time (s) in quadrants andtraveled distance (cm) in the four quadrants. The probe trial providesadditional information about how well an animal learned the position ofthe escape platform. If an animal spends more time and swims a longerdistance in the quadrant where the platform had been positioned duringthe acquisition sessions than in any other quadrant, one concludes thatthe platform position has been learned well.

[0348] In order to assess the effects of putative cognition enhancingcompounds, rats or mice with specific brain lesions which impaircognitive functions, or animals treated with compounds such asscopolamine or MK-801, which interfere with normal learning, or agedanimals which suffer from cognitive deficits, are used.

[0349] The T-maze Spontaneous Alternation Task

[0350] The T-maze spontaneous alternation task (TeMCAT) assesses thespatial memory performance in mice. The start arm and the two goal armsof the T-maze are provided with guillotine doors which can be operatedmanually by the experimenter. A mouse is put into the start arm at thebeginning of training. The guillotine door is closed. In the firsttrial, the ‘forced trial’, either the left or right goal arm is blockedby lowering the guillotine door. After the mouse has been released fromthe start arm, it will negotiate the maze, eventually enter the opengoal arm, and return to the start position, where it will be confinedfor 5 seconds, by lowering the guillotine door. Then, the animal canchoose freely between the left and right goal arm (all guillotine-doorsopened) during 14 ‘free choice’ trials. As soon a the mouse has enteredone goal arm, the other one is closed. The mouse eventually returns tothe start arm and is free to visit whichever go alarm it wants afterhaving been confined to the start arm for 5 seconds. After completion of14 free choice trials in one session, the animal is removed from themaze. During training, the animal is never handled.

[0351] The percent alternations out of 14 trials is calculated. Thispercentage and the total time needed to complete the first forced trialand the subsequent 14 free choice trials (in s) is analyzed. Cognitivedeficits are usually induced by an injection of scopolamine, 30 minbefore the start of the training session. Scopolamine reduced theper-cent alternations to chance level, or below. A cognition enhancer,which is always administered before the training session, will at leastpartially, antagonize the scopolamine-induced reduction in thespontaneous alternation rate.

EXAMPLE 8

[0352] Proliferation Inhibition Assay: Antisense OligonucleotidesSuppress the Growth of Cancer Cell Lines

[0353] The cell line used for testing is the human colon cancer cellline HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calfserum at a concentration of 10,000 cells per milliliter in a volume of0.5 ml and kept at 37° C. in a 95% air/5%CO₂ atmosphere.

[0354] Phosphorothioate oligoribonucleotides are synthesized on anApplied Biosystems Model 380B DNA synthesizer using phosphoroamiditechemistry. A sequence of 24 bases complementary to the nucleotides atposition 1 to 24 of SEQ ID NO:1 or SEQ ID NO:16 is used as the testoligonucleotide. As a control, another (random) sequence is used: 5′-TCAACT GAC TAG ATG TAC ATG GAC-3′. Following assembly and deprotection,oligonucleotides are ethanol-precipitated twice, dried, and suspended inphosphate buffered saline at the desired concentration. Purity of theoligonucleotides is tested by capillary gel electrophoresis and ionexchange HPLC. The purified oligonucleotides are added to the culturemedium at a concentration of 10 μM once per day for seven days.

[0355] The addition of the test oligonucleotide for seven days resultsin significantly reduced expression of human serine/threonine proteinkinase as determined by Western blotting. This effect is not observedwith the control oligonucleotide. After 3 to 7 days, the number of cellsin the cultures is counted using an automatic cell counter. The numberof cells in cultures treated with the test oligonucleotide (expressed as100%) is compared with the number of cells in cultures treated with thecontrol oligonucleotide. The number of cells in cultures treated withthe test oligonucleotide is not more than 30% of control, indicatingthat the inhibition of human serine/threonine protein kinase has ananti-proliferative effect on cancer cells.

EXAMPLE 9

[0356] In Vivo Testing of Compounds/Target Validation

[0357] 1. Acute Mechanistic Assays

[0358] 1.1. Reduction in Mitogenic Plasma Hormone Levels

[0359] This non-tumor assay measures the ability of a compound to reduceeither the endogenous level of a circulating hormone or the level ofhormone produced in response to a biologic stimulus. Rodents areadministered test compound (p.o., i.p., i.v., i.m., or s.c.). At apredetermined time after administration of test compound, blood plasmais collected. Plasma is assayed for levels of the hormone of interest.If the normal circulating levels of the hormone are too low and/orvariable to provide consistent results, the level of the hormone may beelevated by a pre-treatment with a biologic stimulus (i.e., LHRH may beinjected i.m. into mice at a dosage of 30 ng/mouse to induce a burst oftestosterone synthesis). The timing of plasma collection would beadjusted to coincide with the peak of the induced hormone response.Compound effects are compared to a vehicle-treated control group. AnF-test is preformed to determine if the variance is equal or unequalfollowed by a Student's t-test. Significance is p value≦0.05 compared tothe vehicle control group.

[0360] 1.2. Hollow Fiber Mechanism of Action Assay

[0361] Hollow fibers are prepared with desired cell line(s) andimplanted intraperitoneally and/or subcutaneously in rodents. Compoundsare administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol, these may includeassays for gene expression (bDNA, PCR, or Taqman), or a specificbiochemical activity (i.e., cAMP levels. Results are analyzed byStudent's t-test or Rank Sum test after the variance between groups iscompared by an F-test, with significance at p≦0.05 as compared to thevehicle control group.

[0362] 2. Subacute Functional In Vivo Assays

[0363] 2.1. Reduction in Mass of Hormone Dependent Tissues

[0364] This is another non-tumor assay that measures the ability of acompound to reduce the mass of a hormone dependent tissue (i.e., seminalvesicles in males and uteri in females). Rodents are administered testcompound (p.o., i.p., i.v., i.m., or s.c.) according to a predeterminedschedule and for a predetermined duration (i.e., 1 week). At terminationof the study, animals are weighed, the target organ is excised, anyfluid is expressed, and the weight of the organ is recorded. Bloodplasma may also be collected. Plasma may be assayed for levels of ahormone of interest or for levels of test agent. Organ weights may bedirectly compared or they may be normalized for the body weight of theanimal. Compound effects are compared to a vehicle-treated controlgroup. An F-test is preformed to determine if the variance is equal orunequal followed by a Student's t-test. Significance is p value≦0.05compared to the vehicle control group.

[0365] 2.2. Hollow Fiber Proliferation Assay

[0366] Hollow fibers are prepared with desired cell line(s) andimplanted intraperitoneally and/or subcutaneously in rodents. Compoundsare administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol. Cell proliferation isdetermined by measuring a marker of cell number (i.e., MTT or LDH). Thecell number and change in cell number from the starting inoculum areanalyzed by Student's t-test or Rank Sum test after the variance betweengroups is compared by an F-test, with significance at p≦0.05 as comparedto the vehicle control group.

[0367] 2.3. Anti-angiogenesis Models

[0368] 2.3.1. Corneal Angiogenesis

[0369] Hydron pellets with or without growth factors or cells areimplanted into a micropocket surgically created in the rodent cornea.Compound administration may be systemic or local (compound mixed withgrowth factors in the hydron pellet). Corneas are harvested at 7 dayspost implantation immediately following intracardiac infusion ofcolloidal carbon and are fixed in 10% formalin. Readout is qualitativescoring and/or image analysis. Qualitative scores are compared by RankSum test. Image analysis data is evaluated by measuring the area ofneovascularization (in pixels) and group averages are compared byStudent's t-test (2 tail). Significance is p≦0.005 as compared to thegrowth factor or cells only group.

[0370] 2.3.2. Matrigel Angiogenesis

[0371] Matrigel, containing cells or growth factors, is injectedsubcutaneously. Compounds are administered p.o., i.p., i.v., i.m., ors.c. Matrigel plugs are harvested at predetermined time point(s) andprepared for readout. Readout is an ELISA-based assay for hemoglobinconcentration and/or histological examination (i.e. vessel count,special staining for endothelial surface markers: CD31, factor-8).Readouts are analyzed by Student's t-test, after the variance betweengroups is compared by an F-test, with significance determined at p≦0.05as compared to the vehicle control group.

[0372] 3. Primary Antitumor Efficacy

[0373] 3.1. Early Therapy Models

[0374] 3.1.1. Subcutaneous Tumor

[0375] Tumor cells or fragments are implanted subcutaneously on Day 0.Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., ors.c. according to a predetermined schedule starting at a time, usuallyon Day 1, prior to the ability to measure the tumor burden. Body weightsand tumor measurements are recorded 2-3 times weekly. Mean net body andtumor weights are calculated for each data collection day. Antitumorefficacy may be initially determined by comparing the size of treated(T) and control (C) tumors on a given day by a Student's t-test, afterthe variance between groups is compared by an F-test, with significancedetermined at p≦0.05. The experiment may also be continued past the endof dosing in which case tumor measurements would continue to be recordedto monitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p≦0.05.

[0376]3.1.2. Intraperitoneal/Intracranial Tumor Models

[0377] Tumor cells are injected intraperitoneally or intracranially onDay 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule starting on Day 1. Observations ofmorbidity and/or mortality are recorded twice daily. Body weights aremeasured and recorded twice weekly. Morbidity/mortality data isexpressed in terms of the median time of survival and the number oflongterm survivors is indicated separately. Survival times are used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment.

[0378] 3.2. Established Disease Model

[0379] Tumor cells or fragments are implanted subcutaneously and grownto the desired size for treatment to begin. Once at the predeterminedsize range, mice are randomized into treatment groups. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. according to apredetermined schedule. Tumor and body weights are measured and recorded2-3 times weekly. Mean tumor weights of all groups over days postinoculation are graphed for comparison. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group.

[0380] 3.3. Orthotopic Disease Models

[0381] 3.3.1. Mammary Fat Pad Assay

[0382] Tumor cells or fragments, of mammary adenocarcinoma origin, areimplanted directly into a surgically exposed and reflected mammary fatpad in rodents. The fat pad is placed back in its original position andthe surgical site is closed. Hormones may also be administered to therodents to support the growth of the tumors. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.Tumor and body weights are measured and recorded 2-3 times weekly. Meantumor weights of all groups over days post inoculation are graphed forcomparison. An F-test is preformed to determine if the variance is equalor unequal followed by a Student's t-test to compare tumor sizes in thetreated and control groups at the end of treatment. Significance isp≦0.05 as compared to the control group.

[0383] Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group. In addition, this model provides an opportunity toincrease the rate of spontaneous metastasis of this type of tumor.Metastasis can be assessed at termination of the study by counting thenumber of visible foci per target organ, or measuring the target organweight. The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

[0384] 3.3.2. Intraprostatic Assay

[0385] Tumor cells or fragments, of prostatic adenocarcinoma origin, areimplanted directly into a surgically exposed dorsal lobe of the prostatein rodents. The prostate is externalized through an abdominal incisionso that the tumor can be implanted specifically in the dorsal lobe whileverifying that the implant does not enter the seminal vesicles. Thesuccessfully inoculated prostate is replaced in the abdomen and theincisions through the abdomen and skin are closed. Hormones may also beadministered to the rodents to support the growth of the tumors.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., thelungs), or measuring the target organ weight (i.e., the regional lymphnodes). The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

[0386] 3.3.3. Intrabronchial Assay

[0387] Tumor cells of pulmonary origin may be implanted intrabronchiallyby making an incision through the skin and exposing the trachea. Thetrachea is pierced with the beveled end of a 25 gauge needle and thetumor cells are inoculated into the main bronchus using a flat-ended 27gauge needle with a 90° bend. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Body weightsare measured and recorded 2-3 times weekly. At a predetermined time, theexperiment is terminated and the animal is dissected. The size of theprimary tumor is measured in three dimensions using either a caliper oran ocular micrometer attached to a dissecting scope. An F-test ispreformed to determine if the variance is equal or unequal followed by aStudent's t-test to compare tumor sizes in the treated and controlgroups at the end of treatment. Significance is p≦0.05 as compared tothe control group. This model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ (i.e., the contralateral lung), or measuring thetarget organ weight. The means of these endpoints are compared byStudent's t-test after conducting an F-test, with significancedetermined at p≦0.05 compared to the control group in the experiment.

[0388] 3.3.4. Intracecal Assay

[0389] Tumor cells of gastrointestinal origin may be implantedintracecally by making an abdominal incision through the skin andexternalizing the intestine. Tumor cells are inoculated into the cecalwall without penetrating the lumen of the intestine using a 27 or 30gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule. Body weights are measured andrecorded 2-3 times weekly. At a predetermined time, the experiment isterminated and the animal is dissected. The size of the primary tumor ismeasured in three dimensions using either a caliper or an ocularmicrometer attached to a dissecting scope. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. This model provides an opportunity to increase the rate ofspontaneous metastasis of this type of tumor. Metastasis can be assessedat termination of the study by counting the number of visible foci pertarget organ (i.e., the liver), or measuring the target organ weight.The means of these endpoints are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment.

[0390] 4. Secondary (Metastatic) Antitumor Efficacy

[0391] 4.1. Spontaneous Metastasis

[0392] Tumor cells are inoculated s.c. and the tumors allowed to grow toa predetermined range for spontaneous metastasis studies to the lung orliver. These primary tumors are then excised. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedulewhich may include the period leading up to the excision of the primarytumor to evaluate therapies directed at inhibiting the early stages oftumor metastasis. Observations of morbidity and/or mortality arerecorded daily. Body weights are measured and recorded twice weekly.Potential endpoints include survival time, numbers of visible foci pertarget organ, or target organ weight. When survival time is used as theendpoint the other values are not determined. Survival data is used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment. The mean number ofvisible tumor foci, as determined under a dissecting microscope, and themean target organ weights are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment for both of these endpoints.

[0393] 4.2. Forced Metastasis

[0394] Tumor cells are injected into the tail vein, portal vein, or theleft ventricle of the heart in experimental (forced) lung, liver, andbone metastasis studies, respectively. Compounds are administered p.o.,i.p., i.v., i.m., or s.c. according to a predetermined schedule.Observations of morbidity and/or mortality are recorded daily. Bodyweights are measured and recorded twice weekly. Potential endpointsinclude survival time, numbers of visible foci per target organ, ortarget organ weight. When survival time is used as the endpoint theother values are not determined. Survival data is used to generateKaplan-Meier curves. Significance is p≦0.05 by a log-rank test comparedto the control group in the experiment. The mean number of visible tumorfoci, as determined under a dissecting microscope, and the mean targetorgan weights are compared by Student's t-test after conducting anF-test, with significance at p≦0.05 compared to the vehicle controlgroup in the experiment for both endpoints.

EXAMPLE 10

[0395] Diabetes: In Vivo Testing of Compounds/Target Validation

[0396] 1. Glucose Production:

[0397] Over-production of glucose by the liver, due to an enhanced rateof gluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

[0398] 2. Insulin Sensitivity:

[0399] Both ob/ob and db/db nice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

[0400] 3. Insulin Secretion:

[0401] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, compounds are administered by different routes (p.o.,i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At theappropriate time an intravenous glucose load (0.4 g/kg) is given, bloodis collected one minute later. Plasma insulin levels are determined.Compounds that enhance insulin secretion will increase plasma insulinlevels compared to animals given only glucose. When measuring glucosedisappearance, animals are bled at the appropriate time after compoundadministration, then given either an oral or intraperitoneal glucoseload (1 g/kg), bled again after 15, 30, 60 and 90 minutes and plasmaglucose levels determined. Compounds that increase insulin levels willdecrease glucose levels and the area-under-the glucose curve whencompared to the vehicle-treated group given only glucose.

[0402] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, test compounds which regulate serine/threonine proteinkinase are administered by different routes (p.o., i.p., s.c., or i.v.)to overnight fasted normal rats or mice. At the appropriate time anintravenous glucose load (0.4 g/kg) is given, blood is collected oneminute later. Plasma insulin levels are determined. Test compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60, and 90 minutes and plasma glucose levelsdetermined. Test compounds that increase insulin levels will decreaseglucose levels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

[0403] 4. Glucose Production:

[0404] Over-production of glucose by the liver, due to an enhanced rateof gluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

[0405] 5. Insulin Sensitivity:

[0406] Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

[0407] 6. Insulin Secretion:

[0408] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, compounds are administered by different routes (p.o.,i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At theappropriate time an intravenous glucose load (0.4 g/kg) is given, bloodis collected one minute later. Plasma insulin levels are determined.Compounds that enhance insulin secretion will increase plasma insulinlevels compared to animals given only glucose. When measuring glucosedisappearance, animals are bled at the appropriate time after compoundadministration, then given either an oral or intraperitoneal glucoseload (1 g/kg), bled again after 15, 30, 60 and 90 minutes and plasmaglucose levels determined. Compounds that increase insulin levels willdecrease glucose levels and the area-under-the glucose curve whencompared to the vehicle-treated group given only glucose.

EXAMPLE 11

[0409] Quantitative RT-PCR Analysis of Cancer Tissues and Obesity andDiabetes Tissues. RNA Extraction and cDNA Preparation

[0410] RNA used for Taqman quantitative analysis were either purchased(Clontech, CA) or extracted from tissues using TRIzol reagent (LifeTechnologies, MD) according to a modified vendor protocol which utilizesthe RNeasy protocol (Qiagen, CA).

[0411] One hundred μg of each RNA were treated with DNase I using RNasefree- DNase (Qiagen, CA) for use with RNeasy or QiaAmp columns.

[0412] After elution and quantitation with Ribogreen (Molecular ProbesInc., OR) each sample was reverse transcribed using the GibcoBRLSuperscript II First Strand Synthesis System for RT-PCR according tovendor protocol (Life Technologies, MD). The final concentration of RNAin the reaction mix was 50 ng/μL. Reverse transcription was performedwith 0.5 μg of Oligo dT primer for the cancer panel and 50 ng of RandomHexamers for the obesity and diabetes panel.

[0413] TaqMan Quantitative Analysis

[0414] Specific primers and probe were designed according to PE AppliedBiosystems recommendations and are listed below: forward primer:5′-(CAGCAGTATCGGCCTTGTGTAG)-3′ (SEQ ID NO:19) reverse primer:5′-(GCAGAGGCCAGGGAGTTTATT)-3′ (SEQ ID NO:20) probe: SYBR Green

[0415] The expected length of the PCR product was 105 bp.

[0416] Quantitation experiments were performed on 25 ng of reversetranscribed RNA from each sample. Each determination was done induplicate. 18S ribosomal RNA was measured as a control using thePre-Developed TaqMan Assay Reagents (PDAR)(PE Applied Biosystems, CA).Assay reaction mix was as follows: final TaqMan SYBR Green PCR MasterMix (2x)  1x (PB Applied Biosystems, CA) Forward primer (SEQ ID NO:19)300 nM Reverse primer (SEQ ID NO:20) 300 nM cDNA  25 ng Water to 25 μL18s control: Taqman Universal PCR Master Mix (2x)  1x (PB AppliedBiosystems, CA) PDAR control - 18S RNA (20x)  1x 18S ribosomal forwardprimer 300 nM 18S ribosomal reverse primer 300 nM cDNA  25 ng Water to25 μl

[0417] PCR conditions:

[0418] Once: 2 minutes at 50° C.

[0419] 10 minutes at 95° C. 40 cycles: 15 sec. at 95° C. 1 minute at 60°C.

[0420] The experiment was performed on an ABI Prism 7700 SequenceDetector (PE Applied Biosystems, CA). At the end of the run,fluorescence data acquired during PCR were processed as described in theABI Prism 7700 user's manual.

[0421] For cancer tissues, fold change was calculated using thedelta-delta C_(T) method with normalization to the 18S RNA values.Results are shown in FIG. 22.

[0422] For obesity and diabetes tissues, relative expression wasdetermined as follows. Ct values were normalized to 18S RNA values. Thehighest expressing tissue was then assigned a value of 100. Expressionlevels for the remaining tissues were then expressed as percentages ofthe highest expressing tissue ([ΔC_(T) of tissue x/ΔC_(T) of highestexpresser] X100). Results are shown in FIGS. 23a and b.

EXAMPLE 12

[0423] Treatment of COPD in an Animal Model

[0424] Guinea pigs are exposed on a single occasion to tobacco smoke for50 minutes. Animals are sacrificed between 10 minutes and 24 hourfollowing the end of the exposure and their lungs placed in RNAlater™.The lung tissue is homogenized and total RNA is extracted using aQiagens RNeasy™ Maxi kit. Molecular Probes RiboGreen™ RNA quantitationmethod is used to quantify the amount of RNA in each sample. Total RNAis reverse transcribed and the resultant cDNA was used in a real-timepolymerase chain reaction (PCR). The cDNA is added to a solutioncontaining the sense and anti-sense primers and the6-carboxy-tetramethyl-rhodamine labeled probe of the humanserine-threonine protein kinase gene. Cyclophilin is used as thehousekeeping gene. The expression of the human serine-threonine proteinkinase mRNA is measured using the TaqMan real-time PCR system thatgenerates an amplification curve for each sample. From this curve athreshold cycle value is calculated: the fractional cycle number atwhich the amount of amplified target reaches a fixed threshold. A samplecontaining many copies of the human serine-threonine protein kinase mRNAwill reach this threshold earlier than a sample containing fewer copies.The threshold is set at 0.2 and the threshold cycle C_(T) is calculatedfrom the amplification curve. The C_(T) value for the humanserine-threonine protein kinase mRNA is normalized using the C_(T) valuefor the housekeeping gene.

[0425] Test compounds are evaluated as follows. Animals are pre-treatedwith a test compound between 5 minutes and 1 hour prior to the tobaccosmoke exposure and they are then sacrificed up to 3 hours after thetobacco smoke exposure has been completed. Control animals arepre-treated with the vehicle of the test compound via the route ofadministration chosen for the test compound. A test compound thatreduces the tobacco smoke induced upregulation of the humanserine-threonine protein kinase mRNA or the activity of the proteinrelative to the expression or activity seen in vehicle treated tobaccosmoke exposed animals is identified as an inhibitor of the humanserine-threonine protein kinase activity.

1 20 1 2581 DNA Homo sapiens CDS (113)..(1741) 1 ggagatgcgc gcaaccgcgggagcagccaa gtggactgga ctcttttctt gacttagcta 60 ccaggagcta gagatgctgttgttctatcg tatgtgagaa gtcggcccag ag atg gaa 118 Met Glu 1 aac ttt attctg tat gag gag atc gga aga gga agc aag act gtt gtc 166 Asn Phe Ile LeuTyr Glu Glu Ile Gly Arg Gly Ser Lys Thr Val Val 5 10 15 tat aaa ggg cgacgg aag gga aca atc aat ttt gta gcc att ctt tgt 214 Tyr Lys Gly Arg ArgLys Gly Thr Ile Asn Phe Val Ala Ile Leu Cys 20 25 30 act gat aag tgc aaaagg cct gaa ata acc aac tgg gtc cgt ctc acc 262 Thr Asp Lys Cys Lys ArgPro Glu Ile Thr Asn Trp Val Arg Leu Thr 35 40 45 50 cgt gaa ata aaa cacaag aat att gta act ttt cat gaa tgg tat gaa 310 Arg Glu Ile Lys His LysAsn Ile Val Thr Phe His Glu Trp Tyr Glu 55 60 65 aca agc aac cac ctc tggcta gtg gtg gaa ctc tgc aca ggt ggt tcc 358 Thr Ser Asn His Leu Trp LeuVal Val Glu Leu Cys Thr Gly Gly Ser 70 75 80 tta aaa aca gtt att gct caagat gaa aac ctc cca gaa gat gtt gtg 406 Leu Lys Thr Val Ile Ala Gln AspGlu Asn Leu Pro Glu Asp Val Val 85 90 95 aga gaa ttt gga att gac ctg attagt gga tta cat cat ctt cat aaa 454 Arg Glu Phe Gly Ile Asp Leu Ile SerGly Leu His His Leu His Lys 100 105 110 ctt ggc att ctc ttt tgt gac atttct cct agg aag ata ctc ttg gaa 502 Leu Gly Ile Leu Phe Cys Asp Ile SerPro Arg Lys Ile Leu Leu Glu 115 120 125 130 ggg cct ggc aca ctg aag tttagc aac ttt tgc ttg gca aaa gtg gaa 550 Gly Pro Gly Thr Leu Lys Phe SerAsn Phe Cys Leu Ala Lys Val Glu 135 140 145 ggt gaa aat ttg gaa gag ttcttt gct ttg gtg gca gca gag gaa gga 598 Gly Glu Asn Leu Glu Glu Phe PheAla Leu Val Ala Ala Glu Glu Gly 150 155 160 gga ggt gat aat ggg gaa aatgtc ctg aag aaa agc atg aaa agt aga 646 Gly Gly Asp Asn Gly Glu Asn ValLeu Lys Lys Ser Met Lys Ser Arg 165 170 175 gtc aaa gga tct cct gta tatacg gca cca gaa gtt gtg agg ggt gct 694 Val Lys Gly Ser Pro Val Tyr ThrAla Pro Glu Val Val Arg Gly Ala 180 185 190 gac ttt tcc atc tcc agt gacctc tgg tct ttg ggc tgt ctg ctt tat 742 Asp Phe Ser Ile Ser Ser Asp LeuTrp Ser Leu Gly Cys Leu Leu Tyr 195 200 205 210 gaa atg ttt tca gga aaacct cca ttc ttc tca gaa agt att tca gaa 790 Glu Met Phe Ser Gly Lys ProPro Phe Phe Ser Glu Ser Ile Ser Glu 215 220 225 tta act gaa aag atc ttatgt gaa gat cct ttg cca cct att ccg aaa 838 Leu Thr Glu Lys Ile Leu CysGlu Asp Pro Leu Pro Pro Ile Pro Lys 230 235 240 gat tct tct cgt cct aaagct tct tca gat ttt att aat ttg ctt gat 886 Asp Ser Ser Arg Pro Lys AlaSer Ser Asp Phe Ile Asn Leu Leu Asp 245 250 255 ggg tta ctt caa aga gatcct cag aaa aga ttg act tgg aca agg cta 934 Gly Leu Leu Gln Arg Asp ProGln Lys Arg Leu Thr Trp Thr Arg Leu 260 265 270 ctg cag cat tca ttt tggaag aaa gct ttt gct gga gca gat cag gaa 982 Leu Gln His Ser Phe Trp LysLys Ala Phe Ala Gly Ala Asp Gln Glu 275 280 285 290 tca agc gtc gaa gatctc agt ctc agc aga aac act atg gag tgt tct 1030 Ser Ser Val Glu Asp LeuSer Leu Ser Arg Asn Thr Met Glu Cys Ser 295 300 305 ggg cca caa gat tccaag gag ctt ttg cag aac tct cag agt aga caa 1078 Gly Pro Gln Asp Ser LysGlu Leu Leu Gln Asn Ser Gln Ser Arg Gln 310 315 320 gca aaa ggg cac aagagt ggt caa cca cta ggt cac tct ttc aga cta 1126 Ala Lys Gly His Lys SerGly Gln Pro Leu Gly His Ser Phe Arg Leu 325 330 335 gaa aat cca act gagttt cgg cct aag ggt act ctt gag ggt caa ttg 1174 Glu Asn Pro Thr Glu PheArg Pro Lys Gly Thr Leu Glu Gly Gln Leu 340 345 350 aat gaa tcc atg tttctt ctc agt tct cgt cct act ccc aga act agc 1222 Asn Glu Ser Met Phe LeuLeu Ser Ser Arg Pro Thr Pro Arg Thr Ser 355 360 365 370 act gca gtg gaagta agt cct ggt gag gat atg act cac tgt tca cca 1270 Thr Ala Val Glu ValSer Pro Gly Glu Asp Met Thr His Cys Ser Pro 375 380 385 cag gag act tctcct ctg acc aag att aca agt gga cac ctg agt cag 1318 Gln Glu Thr Ser ProLeu Thr Lys Ile Thr Ser Gly His Leu Ser Gln 390 395 400 cag gac ctg gaatcc cag atg aga gag ctt atc tac acg gac tca gat 1366 Gln Asp Leu Glu SerGln Met Arg Glu Leu Ile Tyr Thr Asp Ser Asp 405 410 415 ctt gtt gtc accccc att atc gac aat cca aag ata atg aaa cag cca 1414 Leu Val Val Thr ProIle Ile Asp Asn Pro Lys Ile Met Lys Gln Pro 420 425 430 cca gtt aaa tttgat gca aaa ata ttg cat cta cca aca tat tca gtg 1462 Pro Val Lys Phe AspAla Lys Ile Leu His Leu Pro Thr Tyr Ser Val 435 440 445 450 gat aag ttatta ttt ctg aaa gat caa gat tgg aat gac ttt ttg caa 1510 Asp Lys Leu LeuPhe Leu Lys Asp Gln Asp Trp Asn Asp Phe Leu Gln 455 460 465 caa gtg tgctcg cag atc gac tcc act gag aag agc atg ggg gcc tcc 1558 Gln Val Cys SerGln Ile Asp Ser Thr Glu Lys Ser Met Gly Ala Ser 470 475 480 cga gcc aagctg aat ctt cct ttg cta ttt gtg cgt ggt ggc tgg tca 1606 Arg Ala Lys LeuAsn Leu Pro Leu Leu Phe Val Arg Gly Gly Trp Ser 485 490 495 cca gga ggtggc cac cag gct cct cca ttc ccc cct gtt cca att gct 1654 Pro Gly Gly GlyHis Gln Ala Pro Pro Phe Pro Pro Val Pro Ile Ala 500 505 510 aat cca gcattt gcg gat agc tcc aaa ctg gga tat acg ggc caa ggt 1702 Asn Pro Ala PheAla Asp Ser Ser Lys Leu Gly Tyr Thr Gly Gln Gly 515 520 525 530 tgc tcacgt gat tgg ttt act ggc ttc gca cac agc tga gctccaggaa 1751 Cys Ser ArgAsp Trp Phe Thr Gly Phe Ala His Ser 535 540 aatacacctg ttgttgaggcaattgttctc ttaactgaat taattaggga aaacttcagg 1811 aacagcagat taaaacagtgccttttacca acccttgggg agctgatcta tcttgtagcc 1871 acccaggaag aaaaaaaaaagaaccctaga gagtgctggg ctgttccctt ggctgcatac 1931 acagtgctaa tgaggtgccttcgggaaggg gaagagcgtg ttgtgaatca catggcagca 1991 aaaattattg aaaatgtctgtaccaccttt tctgctcagg cccagggctt tattacagga 2051 gaaataggac ccattttgtggtacctattc agacactcca ctgctgattc tcttaggata 2111 acagcagtat cggccttgtgtagaatcact cgccattctc ctactgcctt ccagaatgtt 2171 attgaaaagg tgggactgaacccagtaata aactccctgg cctctgccat ctgcaaagtt 2231 cagcagtaca tgttgaccttattcactgcc atgttgtcct gtgggattca tcttcaaaga 2291 ctaatccaag aaaaggtttgacttagattt tacctgttac tctacattaa aaattgtttt 2351 cttctgcatt ttagtggttccacaagtaat gtcatgtttg tagaattcat tttttatccc 2411 aagaggcctt tttgaactttgccaaacctt tgtaccacag aatgttcatc tgaacatgtt 2471 ccaagagcct tttagtgattaaaatagaaa ttctttaaag gaaaaaaaaa ggagatgcgc 2531 gcaaccgcgg gagcagccaagtggactgga ctcttttctt gacttagcta 2581 2 542 PRT Homo sapiens 2 Met GluAsn Phe Ile Leu Tyr Glu Glu Ile Gly Arg Gly Ser Lys Thr 1 5 10 15 ValVal Tyr Lys Gly Arg Arg Lys Gly Thr Ile Asn Phe Val Ala Ile 20 25 30 LeuCys Thr Asp Lys Cys Lys Arg Pro Glu Ile Thr Asn Trp Val Arg 35 40 45 LeuThr Arg Glu Ile Lys His Lys Asn Ile Val Thr Phe His Glu Trp 50 55 60 TyrGlu Thr Ser Asn His Leu Trp Leu Val Val Glu Leu Cys Thr Gly 65 70 75 80Gly Ser Leu Lys Thr Val Ile Ala Gln Asp Glu Asn Leu Pro Glu Asp 85 90 95Val Val Arg Glu Phe Gly Ile Asp Leu Ile Ser Gly Leu His His Leu 100 105110 His Lys Leu Gly Ile Leu Phe Cys Asp Ile Ser Pro Arg Lys Ile Leu 115120 125 Leu Glu Gly Pro Gly Thr Leu Lys Phe Ser Asn Phe Cys Leu Ala Lys130 135 140 Val Glu Gly Glu Asn Leu Glu Glu Phe Phe Ala Leu Val Ala AlaGlu 145 150 155 160 Glu Gly Gly Gly Asp Asn Gly Glu Asn Val Leu Lys LysSer Met Lys 165 170 175 Ser Arg Val Lys Gly Ser Pro Val Tyr Thr Ala ProGlu Val Val Arg 180 185 190 Gly Ala Asp Phe Ser Ile Ser Ser Asp Leu TrpSer Leu Gly Cys Leu 195 200 205 Leu Tyr Glu Met Phe Ser Gly Lys Pro ProPhe Phe Ser Glu Ser Ile 210 215 220 Ser Glu Leu Thr Glu Lys Ile Leu CysGlu Asp Pro Leu Pro Pro Ile 225 230 235 240 Pro Lys Asp Ser Ser Arg ProLys Ala Ser Ser Asp Phe Ile Asn Leu 245 250 255 Leu Asp Gly Leu Leu GlnArg Asp Pro Gln Lys Arg Leu Thr Trp Thr 260 265 270 Arg Leu Leu Gln HisSer Phe Trp Lys Lys Ala Phe Ala Gly Ala Asp 275 280 285 Gln Glu Ser SerVal Glu Asp Leu Ser Leu Ser Arg Asn Thr Met Glu 290 295 300 Cys Ser GlyPro Gln Asp Ser Lys Glu Leu Leu Gln Asn Ser Gln Ser 305 310 315 320 ArgGln Ala Lys Gly His Lys Ser Gly Gln Pro Leu Gly His Ser Phe 325 330 335Arg Leu Glu Asn Pro Thr Glu Phe Arg Pro Lys Gly Thr Leu Glu Gly 340 345350 Gln Leu Asn Glu Ser Met Phe Leu Leu Ser Ser Arg Pro Thr Pro Arg 355360 365 Thr Ser Thr Ala Val Glu Val Ser Pro Gly Glu Asp Met Thr His Cys370 375 380 Ser Pro Gln Glu Thr Ser Pro Leu Thr Lys Ile Thr Ser Gly HisLeu 385 390 395 400 Ser Gln Gln Asp Leu Glu Ser Gln Met Arg Glu Leu IleTyr Thr Asp 405 410 415 Ser Asp Leu Val Val Thr Pro Ile Ile Asp Asn ProLys Ile Met Lys 420 425 430 Gln Pro Pro Val Lys Phe Asp Ala Lys Ile LeuHis Leu Pro Thr Tyr 435 440 445 Ser Val Asp Lys Leu Leu Phe Leu Lys AspGln Asp Trp Asn Asp Phe 450 455 460 Leu Gln Gln Val Cys Ser Gln Ile AspSer Thr Glu Lys Ser Met Gly 465 470 475 480 Ala Ser Arg Ala Lys Leu AsnLeu Pro Leu Leu Phe Val Arg Gly Gly 485 490 495 Trp Ser Pro Gly Gly GlyHis Gln Ala Pro Pro Phe Pro Pro Val Pro 500 505 510 Ile Ala Asn Pro AlaPhe Ala Asp Ser Ser Lys Leu Gly Tyr Thr Gly 515 520 525 Gln Gly Cys SerArg Asp Trp Phe Thr Gly Phe Ala His Ser 530 535 540 3 1051 PRT Homosapiens 3 Met Glu Pro Gly Arg Gly Gly Val Glu Thr Val Gly Lys Phe GluPhe 1 5 10 15 Ser Arg Lys Asp Leu Ile Gly His Gly Ala Phe Ala Val ValPhe Lys 20 25 30 Gly Arg His Arg Glu Lys His Asp Leu Glu Val Ala Val LysCys Ile 35 40 45 Asn Lys Lys Asn Leu Ala Lys Ser Gln Thr Leu Leu Gly LysGlu Ile 50 55 60 Lys Ile Leu Lys Glu Leu Lys His Glu Asn Ile Val Ala LeuTyr Asp 65 70 75 80 Phe Gln Glu Met Ala Asn Ser Val Tyr Leu Val Met GluTyr Cys Asn 85 90 95 Gly Gly Asp Leu Ala Asp Tyr Leu His Thr Met Arg ThrLeu Ser Glu 100 105 110 Asp Thr Val Arg Leu Phe Leu Gln Gln Ile Ala GlyAla Met Arg Leu 115 120 125 Leu His Ser Lys Gly Ile Ile His Arg Asp LeuLys Pro Gln Asn Ile 130 135 140 Leu Leu Ser Asn Pro Gly Gly Arg Arg AlaAsn Pro Ser Asn Ile Arg 145 150 155 160 Val Lys Ile Ala Asp Phe Gly PheAla Arg Tyr Leu Gln Ser Asn Met 165 170 175 Met Ala Ala Thr Leu Cys GlySer Pro Met Tyr Met Ala Pro Glu Val 180 185 190 Ile Met Ser Gln His TyrAsp Gly Lys Ala Asp Leu Trp Ser Ile Gly 195 200 205 Thr Ile Val Tyr GlnCys Leu Thr Gly Lys Ala Pro Phe Gln Ala Ser 210 215 220 Ser Pro Gln AspLeu Arg Leu Phe Tyr Glu Lys Asn Lys Thr Leu Val 225 230 235 240 Pro AlaIle Pro Arg Glu Thr Ser Ala Pro Leu Arg Gln Leu Leu Leu 245 250 255 AlaLeu Leu Gln Arg Asn His Lys Asp Arg Met Asp Phe Asp Glu Phe 260 265 270Phe His His Pro Phe Leu Asp Ala Ser Thr Pro Ile Lys Lys Ser Pro 275 280285 Pro Val Pro Val Pro Ser Tyr Pro Ser Ser Gly Ser Gly Ser Ser Ser 290295 300 Ser Ser Ser Ser Ala Ser His Leu Ala Ser Pro Pro Ser Leu Gly Glu305 310 315 320 Met Pro Gln Leu Gln Lys Thr Leu Thr Ser Pro Ala Asp AlaAla Gly 325 330 335 Phe Leu Gln Gly Ser Arg Asp Ser Gly Gly Ser Ser LysAsp Ser Cys 340 345 350 Asp Thr Asp Asp Phe Val Met Val Pro Ala Gln PhePro Gly Asp Leu 355 360 365 Val Ala Glu Ala Ala Ser Ala Lys Pro Pro ProAsp Ser Leu Leu Cys 370 375 380 Ser Gly Ser Ser Leu Val Ala Ser Ala GlyLeu Glu Ser His Gly Arg 385 390 395 400 Thr Pro Ser Pro Ser Pro Thr CysSer Ser Ser Pro Ser Pro Ser Gly 405 410 415 Arg Pro Gly Pro Phe Ser SerAsn Arg Tyr Gly Ala Ser Val Pro Ile 420 425 430 Pro Val Pro Thr Gln ValHis Asn Tyr Gln Arg Ile Glu Gln Asn Leu 435 440 445 Gln Ser Pro Thr GlnGln Gln Thr Ala Arg Ser Ser Ala Ile Arg Arg 450 455 460 Ser Gly Ser ThrThr Pro Leu Gly Phe Gly Arg Ala Ser Pro Ser Pro 465 470 475 480 Pro SerHis Thr Asp Gly Ala Met Leu Ala Arg Lys Leu Ser Leu Gly 485 490 495 GlyGly Arg Pro Tyr Thr Pro Ser Pro Gln Val Gly Thr Ile Pro Glu 500 505 510Arg Pro Ser Trp Ser Arg Val Pro Ser Pro Gln Gly Ala Asp Val Arg 515 520525 Val Gly Arg Ser Pro Arg Pro Gly Ser Ser Val Pro Glu His Ser Pro 530535 540 Arg Thr Thr Gly Leu Gly Cys Arg Leu His Ser Ala Pro Asn Leu Ser545 550 555 560 Asp Phe His Val Val Arg Pro Lys Leu Pro Lys Pro Pro ThrAsp Pro 565 570 575 Leu Gly Ala Thr Phe Ser Pro Pro Gln Thr Ser Ala ProGln Pro Cys 580 585 590 Pro Gly Leu Gln Ser Cys Arg Pro Leu Arg Gly SerPro Lys Leu Pro 595 600 605 Asp Phe Leu Gln Arg Ser Pro Leu Pro Pro IleLeu Gly Ser Pro Thr 610 615 620 Lys Ala Gly Pro Ser Phe Asp Phe Pro LysThr Pro Ser Ser Gln Asn 625 630 635 640 Leu Leu Thr Leu Leu Ala Arg GlnGly Val Val Met Thr Pro Pro Arg 645 650 655 Asn Arg Thr Leu Pro Asp LeuSer Glu Ala Ser Pro Phe His Gly Gln 660 665 670 Gln Leu Gly Ser Gly LeuArg Pro Ala Glu Asp Thr Arg Gly Pro Phe 675 680 685 Gly Arg Ser Phe SerThr Ser Arg Ile Thr Asp Leu Leu Leu Lys Ala 690 695 700 Ala Phe Gly ThrGln Ala Ser Asp Ser Gly Ser Thr Asp Ser Leu Gln 705 710 715 720 Glu LysPro Met Glu Ile Ala Pro Ser Ala Gly Phe Gly Gly Thr Leu 725 730 735 HisPro Gly Ala Arg Gly Gly Gly Ala Ser Ser Pro Ala Pro Val Val 740 745 750Phe Thr Val Gly Ser Pro Pro Ser Gly Ala Thr Pro Pro Gln Ser Thr 755 760765 Arg Thr Arg Met Phe Ser Val Gly Ser Ser Ser Ser Leu Gly Ser Thr 770775 780 Gly Ser Ser Ser Ala Arg His Leu Val Pro Gly Ala Cys Gly Glu Ala785 790 795 800 Pro Glu Leu Ser Ala Pro Gly His Cys Cys Ser Leu Ala AspPro Leu 805 810 815 Ala Ala Asn Leu Glu Gly Ala Val Thr Phe Glu Ala ProAsp Leu Pro 820 825 830 Glu Glu Thr Leu Met Glu Gln Glu His Thr Glu ThrLeu His Ser Leu 835 840 845 Arg Phe Thr Leu Ala Phe Ala Gln Gln Val LeuGlu Ile Ala Ala Leu 850 855 860 Lys Gly Ser Ala Ser Glu Ala Ala Gly GlyPro Glu Tyr Gln Leu Gln 865 870 875 880 Glu Ser Val Val Ala Asp Gln IleSer Gln Leu Ser Arg Glu Trp Gly 885 890 895 Phe Ala Glu Gln Leu Val LeuTyr Leu Lys Val Ala Glu Leu Leu Ser 900 905 910 Ser Gly Leu Gln Thr AlaIle Asp Gln Ile Arg Ala Gly Lys Leu Cys 915 920 925 Leu Ser Ser Thr ValLys Gln Val Val Arg Arg Leu Asn Glu Leu Tyr 930 935 940 Lys Ala Ser ValVal Ser Cys Gln Gly Leu Ser Leu Arg Leu Gln Arg 945 950 955 960 Phe PheLeu Asp Lys Gln Arg Leu Leu Asp Gly Ile His Gly Val Thr 965 970 975 AlaGlu Arg Leu Ile Leu Ser His Ala Val Gln Met Val Gln Ser Ala 980 985 990Ala Leu Asp Glu Met Phe Gln His Arg Glu Gly Cys Val Pro Arg Tyr 995 10001005 His Lys Ala Leu Leu Leu Leu Glu Gly Leu Gln His Thr Leu Thr 10101015 1020 Asp Gln Ala Asp Ile Glu Asn Ile Ala Lys Cys Lys Leu Cys Ile1025 1030 1035 Glu Arg Arg Leu Ser Ala Leu Leu Ser Gly Val Tyr Ala 10401045 1050 4 563 DNA Homo sapiens 4 ttgctgatac agttttattt attttgaaatatttgtcagt atctatacat acacaaggca 60 ctgtctaggc actggagtgg agtgttgaacaagacagttc aaaatcccga ttccaatgga 120 gcttgtagtc tcaacaacag gtgtattttcctggagctca gctgtgtgcg aagccagtaa 180 accaatcacg tgagcaacct tggcccgtatatcccagttt ggagctatcc gcaaatgctg 240 gattagcaat tggaacaggg gggaatggaggagcctggtg gccacctcct ggtgaccagc 300 caccacgcac aaatagcaaa ggagattcagcttggctcgg gaggccccca tgctcttctc 360 agtggagtcg atctgcgagc acacttgttgcaaaaagtca ttccaatctt gatctttcag 420 aaataataac ttatccactg aatatgttggtagatgcaat atttttgcat caaatttaac 480 tggtggctgt ttcattatct ttggattgtcgataatgggg gtgacaacaa gatctgagtc 540 cgtgtagata agctctctca tct 563 5 513DNA Homo sapiens 5 tttgctgata cagttttatt tattttgaaa tatttgtcagtatctataca tacacaaggc 60 actgtctagg cactggagtg gagtgttgaa caagacagttcaaaatcccg attccaatgg 120 agcttgtagt ctcaacaaca ggtgtatttt cctggagctcagttgtgtgc gaagccagta 180 aaccaatcac gtgagcaacc ttggcccgta tatcccagtttggagctatc cgcaaatgct 240 ggattagcaa ttggaacagg ggggaatgga ggagcctggtggccacctcc tggtgaccag 300 ccaccacgca caaatagcaa aggagattca gcttggctcgggaggccccc atgctcttct 360 cagtggagtc gatctgcgag cacacttgtt gcaaaaagtcattccaatct tgatctttca 420 gaaataataa cttatccact gaatatgttg gtagatgcaatatttttgca tcaaatttaa 480 ctggtggctg tttcattatc tttggattgt cga 513 6 501DNA Homo sapiens 6 tttgctgatt cagttttatt tattttgaaa tatttgtcagtatctataca tacacaaggc 60 actgtctagg cactggagtg gagtgttgaa caagacagttcaaaatcccg attccaatgg 120 agcttgtagt ctcaacaaca ggtgtatttt cctggagctcagttgtgtgc gaagccagta 180 aaccaatcac gtgagcaacc ttggcccgta tatcccagtttggagctatc cgcaaatgct 240 ggattagcaa ttggaacagg ggggaatgga ggagcctggtggccacctcc tggtgaccag 300 ccaccacgca caaatagcaa aggagattca gcttggctcgggaggccccc atgctcttct 360 cagtggagtc gatctgcgag cacacttgtt gcaaaaagtcattccaatct tgatctttca 420 gaaataataa cttatccact gaatatgttg gtagatgcaatatttttgca tcaaatttaa 480 ctggtggctg tttcattatc t 501 7 487 DNA Homosapiens 7 tttgctgttt tatttttatt tattttgaaa tattgggggg tgtttatacatacacaaggc 60 actgtctagg cactggagtg gagtgttgaa caagacagtt caaaatcccgattccaatgg 120 agcttgtagt ctcaacaaca ggtgtatttt cctggagctc agctgtgtgcgaagccagta 180 aaccaatcac gtgagcaacc ttggcccgta tatcccagtt tggagctatccgcaaatgct 240 ggattagcaa ttggaacagg ggggaatgga ggagcctggt ggccacctcctggtgaccag 300 ccaccacgca caaatagcaa aggagattca gcttggctcg ggaggcccccatgctcttct 360 cagtggagtc gatctgcgag cacacttgtt gcaaaaagtc attccaatcttgatctttca 420 gaaataataa cttatccact gaatatgttg gtagatgcaa tatttttgcatcaaatttaa 480 ctggtgg 487 8 465 DNA Homo sapiens misc_feature(119)..(119) n=a, c, g or t 8 tggacctgtc ctgaggcaga ggccgagatgcgcgcaaccg cgggagcagc caagtggact 60 ggactctttt cttgacttag ctaccaggagctagagatgc tgttattcta tcgtatgtna 120 gaagtcggcc cagagatgga aaactttattctgtatgagg agatcggaag aggaagcaag 180 actgttgtct ataaagggcg acggaagggaacaatcaatt ttgtagccat tctttgtact 240 gataagtgca gaaggcctga aataaccaactgggtccgtc tcacccgtga aataaaacac 300 aagantattg taacttttca tgaatggtatgaaacaagca nccacctctg gctagtggtg 360 gaactctgca caggtcagga ttatggttgattacttccat ggatgtacac atggacaagg 420 tggttcctta aaaacagtta ttgctcaagatgaaaacctc ccaga 465 9 482 DNA Homo sapiens 9 tttgctgatt ttgttttatttattttgaaa tatttgtcag tatctataca tacacaaggc 60 actgtctagg cactggagtggagtgttgaa caagacagtt caaaatcccg attccaatgg 120 agcttgtagt ctcaacaacaggtgtatttt cctggagctc agttgtgtgc gaagccagta 180 aaccaatcac gtgagcaaccttggcccgta tatcccagtt tggagctatc cgcaaatgct 240 ggattagcaa ttggaacaggggggaatgga ggagcctggt gccacctcct ggtgaccagc 300 caccacgcac aaatagcaaaggagattcag cttggctcgg gaggccccca tgctcttctc 360 agtggagtcg atctgcgagcacacttgttg caaaaagtca ttccaatctt gatctttcag 420 aaataataac ttatccactgaatatgttgg tagatgcaat atttttgcat caaatttaac 480 tg 482 10 493 DNA Homosapiens 10 cacgaggtgg atttgggcta agactccagg tagcacctga tctagtggttctcaacattg 60 atggcatgtt ggtgttaact gaagaatccc gaggctccta aatttgaagaggaaaggttg 120 atttcttatg cagcctgcag gcaattgttc tcttaactga attaattagggaaaacttca 180 ggaacagcaa attaaaacag tgccttttac caacccttgg ggagctgatctatcttgtag 240 ccacccagga agaaaaaaaa aagaacccta gagagtgctg ggctgttcccttggctgcat 300 acacagtgct aatgaggtgc cttcgggaag gggaagagcg tgttgtgaatcacatggcag 360 caaaaattat tgaaaatgtc tgtaccacct tttctgctca gtcccagggctttattacag 420 gagaaatagg acccattttg tggtacctat tcagacactc cactgctgattctcttagga 480 taacagcagt atc 493 11 482 DNA Homo sapiens 11 cttcctgggtggctacaaga tagatcagct ccccaagggt tggtaaaagg cactgtttta 60 atttgctgttcctgaagttt tccctaatta attcagttaa gagaacaatt gcacaaggca 120 ctgtctaggcactggagtgg agtgttgaac aagacagttc aaaatcccga ttccaatgga 180 gcttgtagtctcaacaacag gtgtattttc ctggagctca gttgtgtgcg aagccagtaa 240 accaatcacgtgagcaacct tggcccgtat atcccagttt ggagctatcc gcaaatgctg 300 gattagcaattggaacaggg gggaatggag gagcctggtg gccacctcct ggtgaccagc 360 caccacgcacaaatagcaaa ggagattcag cttggctcgg gaggccccca tgctcttctc 420 agtggagtcgatctgcgagc acacttgttg caaaaagtca ttccaatctt gatctttcag 480 aa 482 12 436DNA Homo sapiens 12 tttttttttt tttgctgata cagttttatt tattttgaaatatttgtcag tatctataca 60 tacacaaggc actgtctagg cactggagtg gagtgttgaacaagacagtt caaaatcccg 120 attccaatgg agcttgtagt ctcaacaaca ggtgtattttcctggagctc agttgtgtgc 180 gaacgcagta aaccaatcac gtgagcaacc ttggcccgtatatcccagtt tggagctatc 240 cgcaaatgct ggattagcaa ttggaacagg ggggaatggaggagcctggt ggccacctcc 300 tggtgaccag ccaccacgca caaatagcaa aggagattcagcttggctcg ggaggccccc 360 atgctcttct cagtggagtc gatctgcgag cacacttgttgcaaaaagtc attccaatct 420 tgatctttca gaaata 436 13 404 DNA Homo sapiens13 tttttttaaa tgtttactgg ttaatctagg tgatggaaac aaaggtgatt attacagctt 60tcttttcttt tctaatcttt gaaacatttt ataataatag gctgtagagg attcagagtt 120ccccttcccg aaggcacctc attagcactg tgtatgcagc caagggaaca gcccagcact 180ctctagggtt cttttttttt tcttcctggg gggctacaag atagatcagc tccccaaggg 240ttggtaaaag gcactgtttt aatttgctgt tcctgaagtt ttccctaatt aattcagtta 300agagaacaat tgcctcaaca acaggtgtat tttcctggag ctcagttgtg tgcgaagcca 360gtaaaccaat cacgtgagca accttggccc gtatatccca gttt 404 14 396 DNA Homosapiens 14 tttttttttt tttttttttt ttttgctgat acagttttat ttattttgaaatatttgtca 60 gtatctatac atacacaagg cactgtctag gcactggagg ggagggttgaacaagacagt 120 tcaaaatccc gattccaatg gagcttgtag tctcaacaac aggtgtattttcctggagct 180 cagctgtgtg cgaagccagt aaaccaatca cgtgagcaac cttggcccgtatatcccagt 240 ttggagctat ccgcaaatgc tggattagca attggaacag gggggaatggaggagcctgg 300 tggccacctc ctggtgacca gccaccacgc acaaatagca aaggagattcagcttggctc 360 gggaggcccc catgctcttc tcagtggagt cgatct 396 15 400 DNAHomo sapiens 15 cacgaggtgg atttgggcta agactccagg tagcacctga tctagtggttctcaacattg 60 atggcatgtt ggtgttaact gaagaatccc gaggctccta aatttgaagaggaaaggttg 120 atttcttatg cagcctgcag gcaattgttc tcttaactga attaattagggaaaacttca 180 ggaacagcaa attaaaacag tgccttttac caacccttgg ggagctgatctatcttgtag 240 ccacccagga agaaaaaaaa aagaacccta aagagtgctg ggctgttcccttggctgcat 300 acacagtgct aatgaggtgc cttcgggaag gggaagagcg tgttgtgaatcacatggcag 360 caaaaattat tgaaaatgtc tgtaccacct tttctgctca 400 16 1743DNA Homo sapiens 16 atggaaaact ttattctgta tgaggagatc ggaagaggaagcaagactgt tgtctataaa 60 gggcgacgga agggaacaat caattttgta gccattctttgtactgataa gtgcaaaagg 120 cctgaaataa ccaactgggt ccgtctcacc cgtgaaataaaacacaagaa tattgtaact 180 tttcatgaat ggtatgaaac aagcaaccac ctctggctagtggtggaact ctgcacaggt 240 ggttccttaa aaacagttat tgctcaagat gaaaacctcccagaagatgt tgtgagagaa 300 tttggaattg acctgattag tggattacat catcttcataaacttggcat tctcttttgt 360 gacatttctc ctaggaagat actcttggaa gggcctggcacactgaagtt tagcaacttt 420 tgcttggcaa aagtggaagg tgaaaatttg gaagagttctttgctttggt ggcagcagag 480 gaaggaggag gtgataatgg ggaaaatgtc ctgaagaaaagcatgaaaag tagagtcaaa 540 ggatctcctg tatatacggc accagaagtt gtgaggggtgctgacttttc catctccagt 600 gacctctggt ctttgggctg tctgctttat gaaatgttttcaggaaaacc tccattcttc 660 tcagaaagta tttcagaatt aactgaaaag atcttatgtgaagatccttt gccacctatt 720 ccgaaagatt cttctcgtcc taaagcttct tcagattttattaatttgct tgatgggtta 780 cttcaaagag atcctcagaa aagattgact tggacaaggctactgcagca ttcattttgg 840 aagaaagctt ttgctggagc agatcaggaa tcaagcgtcgaagatctcag tctcagcaga 900 aacactatgg agtgttctgg gccacaagat tccaaggagcttttgcagaa ctctcagagt 960 agacaagcaa aagggcacaa gagtggtcaa ccactaggtcactctttcag actagaaaat 1020 ccaactgagt ttcggcctaa gggtactctt gagggtcaattgaatgaatc catgtttctt 1080 ctcagttctc gtcctactcc cagaactagc actgcagtggaagtaagtcc tggtgaggat 1140 atgactcact gttcaccaca ggagacttct cctctgaccaagattacaag tggacacctg 1200 agtcagcagg acctggaatc ccagatgaga gagcttatctacacggactc agatcttgtt 1260 gtcaccccca ttatcgacaa tccaaagata atgaaacagccaccagttaa atttgatgca 1320 aaaatattgc atctaccaac atattcagtg gataagttattatttctgaa agatcaagat 1380 tggaatgact ttttgcaaca agtgtgctcg cagatcgactccactgagaa gagcatgggg 1440 gcctcccgag ccaagctgaa tctcctttgc tatttgtgcgtggtggctgg tcaccaggag 1500 gtggccacca ggctcctcca ttcccccctg ttccaattgctaatccagca tttgcggata 1560 gctccaaact gggatatacg ggccaaggtt gctcacgtgattggtttact ggcttcgcac 1620 acagctgagc tccaggaaaa tacacctgtt gttgagactacaagctccat tggaatcggg 1680 attttgaact gtcttgttca acactccact ccagtgcctagacagtgcct tgtgtatgta 1740 tag 1743 17 580 PRT Homo sapiens 17 Met GluAsn Phe Ile Leu Tyr Glu Glu Ile Gly Arg Gly Ser Lys Thr 1 5 10 15 ValVal Tyr Lys Gly Arg Arg Lys Gly Thr Ile Asn Phe Val Ala Ile 20 25 30 LeuCys Thr Asp Lys Cys Lys Arg Pro Glu Ile Thr Asn Trp Val Arg 35 40 45 LeuThr Arg Glu Ile Lys His Lys Asn Ile Val Thr Phe His Glu Trp 50 55 60 TyrGlu Thr Ser Asn His Leu Trp Leu Val Val Glu Leu Cys Thr Gly 65 70 75 80Gly Ser Leu Lys Thr Val Ile Ala Gln Asp Glu Asn Leu Pro Glu Asp 85 90 95Val Val Arg Glu Phe Gly Ile Asp Leu Ile Ser Gly Leu His His Leu 100 105110 His Lys Leu Gly Ile Leu Phe Cys Asp Ile Ser Pro Arg Lys Ile Leu 115120 125 Leu Glu Gly Pro Gly Thr Leu Lys Phe Ser Asn Phe Cys Leu Ala Lys130 135 140 Val Glu Gly Glu Asn Leu Glu Glu Phe Phe Ala Leu Val Ala AlaGlu 145 150 155 160 Glu Gly Gly Gly Asp Asn Gly Glu Asn Val Leu Lys LysSer Met Lys 165 170 175 Ser Arg Val Lys Gly Ser Pro Val Tyr Thr Ala ProGlu Val Val Arg 180 185 190 Gly Ala Asp Phe Ser Ile Ser Ser Asp Leu TrpSer Leu Gly Cys Leu 195 200 205 Leu Tyr Glu Met Phe Ser Gly Lys Pro ProPhe Phe Ser Glu Ser Ile 210 215 220 Ser Glu Leu Thr Glu Lys Ile Leu CysGlu Asp Pro Leu Pro Pro Ile 225 230 235 240 Pro Lys Asp Ser Ser Arg ProLys Ala Ser Ser Asp Phe Ile Asn Leu 245 250 255 Leu Asp Gly Leu Leu GlnArg Asp Pro Gln Lys Arg Leu Thr Trp Thr 260 265 270 Arg Leu Leu Gln HisSer Phe Trp Lys Lys Ala Phe Ala Gly Ala Asp 275 280 285 Gln Glu Ser SerVal Glu Asp Leu Ser Leu Ser Arg Asn Thr Met Glu 290 295 300 Cys Ser GlyPro Gln Asp Ser Lys Glu Leu Leu Gln Asn Ser Gln Ser 305 310 315 320 ArgGln Ala Lys Gly His Lys Ser Gly Gln Pro Leu Gly His Ser Phe 325 330 335Arg Leu Glu Asn Pro Thr Glu Phe Arg Pro Lys Gly Thr Leu Glu Gly 340 345350 Gln Leu Asn Glu Ser Met Phe Leu Leu Ser Ser Arg Pro Thr Pro Arg 355360 365 Thr Ser Thr Ala Val Glu Val Ser Pro Gly Glu Asp Met Thr His Cys370 375 380 Ser Pro Gln Glu Thr Ser Pro Leu Thr Lys Ile Thr Ser Gly HisLeu 385 390 395 400 Ser Gln Gln Asp Leu Glu Ser Gln Met Arg Glu Leu IleTyr Thr Asp 405 410 415 Ser Asp Leu Val Val Thr Pro Ile Ile Asp Asn ProLys Ile Met Lys 420 425 430 Gln Pro Pro Val Lys Phe Asp Ala Lys Ile LeuHis Leu Pro Thr Tyr 435 440 445 Ser Val Asp Lys Leu Leu Phe Leu Lys AspGln Asp Trp Asn Asp Phe 450 455 460 Leu Gln Gln Val Cys Ser Gln Ile AspSer Thr Glu Lys Ser Met Gly 465 470 475 480 Ala Ser Arg Ala Lys Leu AsnLeu Leu Cys Tyr Leu Cys Val Val Ala 485 490 495 Gly His Gln Glu Val AlaThr Arg Leu Leu His Ser Pro Leu Phe Gln 500 505 510 Leu Leu Ile Gln HisLeu Arg Ile Ala Pro Asn Trp Asp Ile Arg Ala 515 520 525 Lys Val Ala HisVal Ile Gly Leu Leu Ala Ser His Thr Ala Glu Leu 530 535 540 Gln Glu AsnThr Pro Val Val Glu Thr Thr Ser Ser Ile Gly Ile Gly 545 550 555 560 IleLeu Asn Cys Leu Val Gln His Ser Thr Pro Val Pro Arg Gln Cys 565 570 575Leu Val Tyr Val 580 18 487 DNA Homo sapiens 18 tttgctgttt tatttttatttattttgaaa tattgggggg tgtttataca tacacaaggc 60 actgtctagg cactggagtggagtgttgaa caagacagtt caaaatcccg attccaatgg 120 agcttgtagt ctcaacaacaggtgtatttt cctggagctc agctgtgtgc gaagccagta 180 aaccaatcac gtgagcaaccttggcccgta tatcccagtt tggagctatc cgcaaatgct 240 ggattagcaa ttggaacaggggggaatgga ggagcctggt ggccacctcc tggtgaccag 300 ccaccacgca caaatagcaaaggagattca gcttggctcg ggaggccccc atgctcttct 360 cagtggagtc gatctgcgagcacacttgtt gcaaaaagtc attccaatct tgatctttca 420 gaaataataa cttatccactgaatatgttg gtagatgcaa tatttttgca tcaaatttaa 480 ctggtgg 487 19 22 DNAHomo sapiens misc_feature Primer 19 cagcagtatc ggccttgtgt ag 22 20 21DNA Homo sapiens misc_feature Primer 20 gcagaggcca gggagtttat t 21

1. An isolated polynucleotide encoding a serine-threonine protein kinasepolypeptide and being selected from the group consisting of: a) apolynucleotide encoding a serine-threonine protein kinase polypeptidecomprising an amino acid sequence selected form the group consisting of:amino acid sequences which are at least about 30% identical to the aminoacid sequence shown in SEQ ID NO: 2; and the amino acid sequence shownin SEQ ID NO: 2; amino acid sequences which are at least about 30%identical to the amino acid sequence shown in SEQ ID NO: 17; and theamino acid sequence shown in SEQ ID NO: 17; b) a polynucleotidecomprising the sequence of SEQ ID NO:1 or 16; c) a polynucleotide whichhybridizes under stringent conditions to a polynucleotide specified in(a) and (b); d) a polynucleotide the sequence of which deviates from thepolynucleotide sequences specified in (a) to (c) due to the degenerationof the genetic code; and e) a polynucleotide which represents afragment, derivative or allelic variation of a polynucleotide sequencespecified in (a to (d).
 2. An expression vector containing anypolynucleotide of claim
 1. 3. A host cell containing the expressionvector of claim
 2. 4. A substantially purified serine-threonine proteinkinase polypeptide encoded by a polynucleotide of claim
 1. 5. A methodfor producing a serine-threonine protein kinase polypeptide, wherein themethod comprises the following steps: a) culturing the host cell ofclaim 3 under conditions suitable for the expression of theserine-threonine protein kinase polypeptide; and b) recovering theserine-threonine protein kinase polypeptide from the host cell culture.6. A method for detection of a polynucleotide encoding aserine-threonine protein kinase polypeptide in a biological samplecomprising the following steps: a) hybridizing any polynucleotide ofclaim 1 to a nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting said hybridizationcomplex.
 7. The method of claim 6, wherein before hybridization, thenucleic acid material of the biological sample is amplified.
 8. A methodfor the detection of a polynucleotide of claim 1 or a serine-threonineprotein kinase polypeptide of claim 4 comprising the steps of:contacting a biological sample with a reagent which specificallyinteracts with the polynucleotide or the serine-threonine protein kinasepolypeptide.
 9. A diagnostic kit for conducting the method of any one ofclaims 6 to
 8. 10. A method of screening for agents which decrease theactivity of a serine-threonine protein kinase, comprising the steps of:contacting a test compound with any serine-threonine protein kinasepolypeptide encoded by any polynucleotide of claim 1; detecting bindingof the test compound to the serine-threonine protein kinase polypeptide,wherein a test compound which binds to the polypeptide is identified asa potential therapeutic agent for decreasing the activity of aserine-threonine protein kinase.
 11. A method of screening for agentswhich regulate the activity of a serine-threonine protein kinase,comprising the steps of: contacting a test compound with aserine-threonine protein kinase polypeptide encoded by anypolynucleotide of claim 1; and detecting a serine-threonine proteinkinase activity of the polypeptide, wherein a test compound whichincreases the serine-threonine protein kinase activity is identified asa potential therapeutic agent for increasing the activity of theserine-threonine protein kinase, and wherein a test compound whichdecreases the serine-threonine protein kinase activity of thepolypeptide is identified as a potential therapeutic agent fordecreasing the activity of the serine-threonine protein kinase.
 12. Amethod of screening for agents which decrease the activity of aserine-threonine protein kinase, comprising the steps of: contacting atest compound with any polynucleotide of claim 1 and detecting bindingof the test compound to the polynucleotide, wherein a test compoundwhich binds to the polynucleotide is identified as a potentialtherapeutic agent for decreasing the activity of serine-threonineprotein kinase.
 13. A method of reducing the activity ofserine-threonine protein kinase, comprising the steps of: contacting acell with a reagent which specifically binds to any polynucleotide ofclaim 1 or any serine-threonine protein kinase polypeptide of claim 4,whereby the activity of serine-threonine protein kinase is reduced. 14.A reagent that modulates the activity of a serine-threonine proteinkinase polypeptide or a polynucleotide wherein said reagent isidentified by the method of any of the claim 10 to
 12. 15. Apharmaceutical composition, comprising: the expression vector of claim 2or the reagent of claim 14 and a pharmaceutically acceptable carrier.16. Use of the expression vector of claim 2 or the reagent of claim 14to produce a medicament for modulating the activity of aserine-threonine protein kinase in a disease.
 17. Use of claim 16wherein the disease is cancer, a CNS disorder, diabetes, or COPD.
 18. AcDNA encoding a polypeptide comprising the amino acid sequence shown inSEQ ID NO:2 or
 17. 19. The cDNA of claim 18 which comprises SEQ ID NO:1or
 16. 20. The cDNA of claim 18 which consists of SEQ ID NO:1 or
 16. 21.An expression vector comprising a polynucleotide which encodes apolypeptide comprising the amino acid sequence shown in SEQ ID NO:2 or17.
 22. The expression vector of claim 21 wherein the polynucleotideconsists of SEQ ID NO:1 or
 16. 23. A host cell comprising an expressionvector which encodes a polypeptide comprising the amino acid sequenceshown in SEQ ID NO:2 or
 17. 24. The host cell of claim 23 wherein thepolynucleotide consists of SEQ ID NO:1 or
 16. 25. A purified polypeptidecomprising the amino acid sequence shown in SEQ ID NO:2 or
 17. 26. Thepurified polypeptide of claim 25 which consists of the amino acidsequence shown in SEQ ID NO:2 or
 17. 27. A fusion protein comprising apolypeptide having the amino acid sequence shown in SEQ ID NO:2 or 17.28. A method of producing a polypeptide comprising the amino acidsequence shown in SEQ ID NO:2 or 17, comprising the steps of: culturinga host cell comprising an expression vector which encodes thepolypeptide under conditions whereby the polypeptide is expressed; andisolating the polypeptide.
 29. The method of claim 28 wherein theexpression vector comprises SEQ ID NO:1 or
 16. 30. A method of detectinga coding sequence for a polypeptide comprising the amino acid sequenceshown in SEQ ID NO:2 or 17, comprising the steps of: hybridizing apolynucleotide comprising 11 contiguous nucleotides of SEQ ID NO:1 or 16to nucleic acid material of a biological sample, thereby forming ahybridization complex; and detecting the hybridization complex.
 31. Themethod of claim 30 further comprising the step of amplifying the nucleicacid material before the step of hybridizing.
 32. A kit for detecting acoding sequence for a polypeptide comprising the amino acid sequenceshown in SEQ ID NO:2 or 17, comprising: a polynucleotide comprising 11contiguous nucleotides of SEQ ID NO: 1 or 16; and instructions for themethod of claim
 30. 33. A method of detecting a polypeptide comprisingthe amino acid sequence shown in SEQ ID NO:2 or 17, comprising the stepsof: contacting a biological sample with a reagent that specificallybinds to the polypeptide to form a reagent-polypeptide complex; anddetecting the reagent-polypeptide complex.
 34. The method of claim 33wherein the reagent is an antibody.
 35. A kit for detecting apolypeptide comprising the amino acid sequence shown in SEQ ID NO:2 or17, comprising: an antibody which specifically binds to the polypeptide;and instructions for the method of claim
 33. 36. A method of screeningfor agents which can modulate the activity of a human serine-threonineprotein kinase, comprising the steps of: contacting a test compound witha polypeptide comprising an amino acid sequence selected from the groupconsisting of: (1) amino acid sequences which are at least about 30%identical to the amino acid sequence shown in SEQ ID NO:2 or 17 and (2)the amino acid sequence shown in SEQ ID NO:2 or 17; and detectingbinding of the test compound to the polypeptide, wherein a test compoundwhich binds to the polypeptide is identified as a potential agent forregulating activity of the human serine-threonine protein kinase. 37.The method of claim 36 wherein the step of contacting is in a cell. 38.The method of claim 36 wherein the cell is in vitro.
 39. The method ofclaim 36 wherein the step of contacting is in a cell-free system. 40.The method of claim 36 wherein the polypeptide comprises a detectablelabel.
 41. The method of claim 36 wherein the test compound comprises adetectable label.
 42. The method of claim 36 wherein the test compounddisplaces a labeled ligand which is bound to the polypeptide.
 43. Themethod of claim 36 wherein the polypeptide is bound to a solid support.44. The method of claim 36 wherein the test compound is bound to a solidsupport.
 45. A method of screening for agents which modulate an activityof a human serine-threonine protein kinase, comprising the steps of:contacting a test compound with a polypeptide comprising an amino acidsequence selected from the group consisting of: (1) amino acid sequenceswhich are at least about 30% identical to the amino acid sequence shownin SEQ ID NO:2 or 17 and (2) the amino acid sequence shown in SEQ IDNO:2 or 17; and detecting an activity of the polypeptide, wherein a testcompound which increases the activity of the polypeptide is identifiedas a potential agent for increasing the activity of the humanserine-threonine protein kinase, and wherein a test compound whichdecreases the activity of the polypeptide is identified as a potentialagent for decreasing the activity of the human serine-threonine proteinkinase.
 46. The method of claim 45 wherein the step of contacting is ina cell.
 47. The method of claim 45 wherein the cell is in vitro.
 48. Themethod of claim 45 wherein the step of contacting is in a cell-freesystem.
 49. A method of screening for agents which modulate an activityof a human serine-threonine protein kinase, comprising the steps of:contacting a test compound with a product encoded by a polynucleotidewhich comprises the nucleotide sequence shown in SEQ ID NO:1 or 16; anddetecting binding of the test compound to the product, wherein a testcompound which binds to the product is identified as a potential agentfor regulating the activity of the human serine-threonine proteinkinase.
 50. The method of claim 49 wherein the product is a polypeptide.51. The method of claim 49 wherein the product is RNA.
 52. A method ofreducing activity of a human serine-threonine protein kinase, comprisingthe step of: contacting a cell with a reagent which specifically bindsto a product encoded by a polynucleotide comprising the nucleotidesequence shown in SEQ ID NO:1 or 16, whereby the activity of a humanserine-threonine protein kinase is reduced.
 53. The method of claim 52wherein the product is a polypeptide.
 54. The method of claim 53 whereinthe reagent is an antibody.
 55. The method of claim 52 wherein theproduct is RNA.
 56. The method of claim 55 wherein the reagent is anantisense oligonucleotide.
 57. The method of claim 56 wherein thereagent is a ribozyme.
 58. The method of claim 52 wherein the cell is invitro.
 59. The method of claim 52 wherein the cell is in vivo.
 60. Apharmaceutical composition, comprising: a reagent which specificallybinds to a polypeptide comprising the amino acid sequence shown in SEQID NO:2 or 17; and a pharmaceutically acceptable carrier.
 61. Thepharmaceutical composition of claim 60 wherein the reagent is anantibody.
 62. A pharmaceutical composition, comprising: a reagent whichspecifically binds to a product of a polynucleotide comprising thenucleotide sequence shown in SEQ ID NO:1 or 16; and a pharmaceuticallyacceptable carrier.
 63. The pharmaceutical composition of claim 62wherein the reagent is a ribozyme.
 64. The pharmaceutical composition ofclaim 62 wherein the reagent is an antisense oligonucleotide.
 65. Thepharmaceutical composition of claim 62 wherein the reagent is anantibody.
 66. A pharmaceutical composition, comprising: an expressionvector encoding a polypeptide comprising the amino acid sequence shownin SEQ ID NO:2 or 17; and a pharmaceutically acceptable carrier.
 67. Thepharmaceutical composition of claim 66 wherein the expression vectorcomprises SEQ ID NO:1 or
 16. 68. A method of treating a serine-threonineprotein kinase dysfunction related disease, wherein the disease isselected from cancer, a CNS disorder, diabetes, or COPD comprising thestep of: administering to a patient in need thereof a therapeuticallyeffective dose of a reagent that modulates a function of a humanserine-threonine protein kinase, whereby symptoms of theserine-threonine protein kinase dysfunction related disease areameliorated.
 69. The method of claim 68 wherein the reagent isidentified by the method of claim
 36. 70. The method of claim 68 whereinthe reagent is identified by the method of claim
 45. 71. The method ofclaim 68 wherein the reagent is identified by the method of claim 49.